Csir-Ugc Net

Amino Acids
• Building blocks of polypeptides/proteins

• Amino acids (aa) are linked to each other via peptide bond
• Peptide bond synthesis occurs during RNA synthesis in the ribosome (found in cytosol or ER)
Structural Features of Amino Acids

• All 20 common aa are α-amino acids
○ α-amino acids = have caroxyl group (-COO- ) & amino group (-NH3+) bonded to the same carbon atom (α carbon)
○ Except glycine, for all the common amino acids the carbon is bonded to four different groups: a carboxyl group, an amino group, an
R group, and a hydrogen atom. Since the the α-carbon is bound to different groups, the α-carbon atom is thus a chiral centre.
○ In glycine, the R group is another hydrogen atom.
○ Aa differ from each other in their side chains (or R groups)
Structures of Amino Acids Mnemonic
• Each sulfur-containing aa can link to other sulfur-containing aa through

oxidation of their sulfhydryl bonds to form sulfur-sulfur bonds
• Acidic & basic aa are charged aa
• All aliphatic aa, as well as methionine - a sulfur-containing aa, and
phenylalanine & tryptophan, which are aromatic aa, are non-polar
• All alcohols & amides, as well as cysteine, which is a sulfur-containing
aa, and tyrosine, which is an aromatic aa, are polar aa.
Stereoisomers of Amino Acids
• Stereoisomers are isomers that differ in spatial arrangement of atoms, rather than order of atomic connectivity.
• Except for glycine, the standard amino acids have asymmetric structures and rotate the plane of polarized light; thus, they are
optically active. These molecules cannot be superimposed on their mirror images. Such nonsuperimposable pairs of molecules are called
enantiomers. The asymmetric atom of an optically active molecule is called the chiral centre and the molecule is said to have the
property of chirality.
• D- and L- notation provides a quick shorthand for designating enantiomers.

Special nomenclature has been developed to specify the absolute configuration of the four substituents of asymmetric carbon atoms. The
absolute configurations of simple sugars and amino acids are specified by the D, L system, based on the absolute configuration of the three-
carbon sugar glyceraldehyde, a convention proposed by Emil Fischer in 1891.
Proteins Page 1
• Most naturally occurring sugars are D-, and most naturally occurring amino acids are L-
• The amino acid residues in protein molecules are exclusively L stereoisomers. D-Amino acid residues have been found in only a few,
generally small peptides, including some peptides of bacterial cell walls and certain peptide antibiotics.
• Why are naturally occurring amino acids L-stereoisomers?
○ The formation of stable, repeating substructures in proteins generally requires that their constituent amino acids be of one
stereochemical series. Cells are able to specifically synthesize the L isomers of amino acids because the active sites of enzymes
are asymmetric, causing the reactions they catalyse to be stereospecific.
Amino Acids Can be Classified by R Group
Nonpolar, Aliphatic R Aromatic R Groups Polar, Uncharged R Positively Charged Negatively Charged
Groups Groups (Basic) R Groups (Acidic) R Groups
The R groups in this class The R groups in this class of The R groups of these The amino acids in which The two amino acids
of amino acids are amino acids have aromatic side amino acids are more the R groups have having R groups with a net
nonpolar and chains. They also absorb soluble in water, or significant positive charge negative charge at pH 7.0
hydrophobic. Their side ultraviolet light. This accounts more hydrophilic, than at pH 7.0 are lysine, which are aspartate and
chains tend to cluster for the characteristic strong those of the nonpolar has a second primary glutamate, each of which
together within proteins, absorbance of light by most amino acids, because amino group at the ε has a second carboxyl
stabilizing protein proteins at a wavelength of 280 they contain functional position on its aliphatic group.
structure by means of nm, a property exploited by groups that form chain; arginine, which has
hydrophobic interactions. researchers in the hydrogen bonds with a positively charged
Examples - characterization of proteins. water. guanidinium group; and
alanine, valine, leucine, This class of amino histidine, which has an
and isoleucine, Glycine, acids includes serine, aromatic imidazole group.
Methionine, Proline threonine, cysteine,
asparagine, and
glutamine
Absorption of light by molecules - Lambert-Beer Law
○ The fraction of the incident light absorbed by a solution at a given wavelength is related to the thickness of the absorbing layer
(path length) and the concentration of the absorbing species. These two relationships are combined into the Lambert-Beer law.
○ where I0 is the intensity of the incident light, I is the intensity of the transmitted light, the ratio I/I0 (the inverse of the ratio in
the equation) is the
transmittance, Ɛ is the molar extinction coefficient (liters/mole-centimeter), c is the concentration of the absorbing species (in
moles/liter), and l is the path length of the light-absorbing sample (in cm). The expression log (I0/I) is called the absorbance,
designated A.
○ The molar extinction coefficient varies with the nature of the absorbing compound, the solvent, and the wavelength, and also with
pH if the light-absorbing species is in equilibrium with an ionization state that has different absorbance properties
Proteins Page 2
Uncommon Amino Acids Also Have Important Functions
4-hydroxyproline found in plant cell wall proteins, and in collagen, a fibrous protein of connective tissues
5-hydroxylysine found in collagen, a fibrous protein of connective tissues
6-N- constituent of myosin, a contractile protein of muscle
Methyllysine
Pyrrolysine Found in several archaeal species
Glutathione cellular reducing agent
γ- found in the blood-clotting protein prothrombin and in certain other proteins that bind
carboxyglutamate Ca2+ as part of their biological function
Pyroglutamate Found in Bacteriorhodopsin
Desmosine found in the fibrous protein elastin
Selenocysteine constituent of just a few known proteins in many organisms ( It is introduced during
protein synthesis rather than created through a post-synthesis modification. It contains
selenium rather than the sulfur of cysteine.)
Ornithine Key intermediates (metabolites) in the biosynthesis of arginine
Citrulline Key intermediates (metabolites) in the urea cycle
GABA, Act as Neurotransmitters & Hormones
Epinephrine,
histamine,
serotonin
Phosphorylated Important in activation and inhibition of enzymatic or signalling activity (Tyr, Ser, Thr
Amino Acids hydroxyl groups can be phosphorylated)
Amino Acids Can Act as Acids and Bases
• Molecules such as amino acids, which bear charged groups of opposite polarity, are known as dipolar ions or zwitterions. Amino acids,
like other ionic compounds, are more soluble in polar solvents than in nonpolar solvents.
• When an amino acid lacking an ionizable R group is dissolved in water at neutral pH, it exists in solution as the dipolar ion , or zwitterion
(German for “hybrid ion”), which can act as either an acid or a base.
• Substances having this dual (acid-base) nature are amphoteric and are often called ampholytes (from “amphoteric electrolytes”).
• A simple monoamino monocarboxylic -amino acid, such as alanine, is a diprotic acid when fully protonated; it has two groups, the —COOH
group and the —NH3 group, that can yield protons
Proteins Page 3
Amino Acids Have Characteristic Titration Curves
• Titration curve of an amino acid is calculated using the Henderson Hesselbech equation Isoelectric point (pI) is the pH at which the molecule has a net
charge = 0 (average of the two appropriate pKa values)
Key Equation: pI = ½(pKi + pKj)
• The pKa is a measure of the tendency of a group to give up a proton, with that tendency decreasing tenfold as the pKa increases by one unit.)
The two ionizable groups of glycine, the carboxyl group and the
amino group, are titrated with a strong base such as NaOH. The plot has two distinct
stages, corresponding to deprotonation of first the carboxylic (-COOH ) group of glycine
followed by the deprotonation of the amine (NH3+) group of glycine.
What information do we get from titration curves?
• The titration curve of glycine gives a quantitative measure of the pKa of each of
the two ionizing groups: 2.34 for the -COOH groupand 9.60 for the -NH3+ group.
• The second piece of information provided by the titration curve of glycine is that
this amino acid has two regions of buffering power. These are the relatively flat
portion of the curve, extending for approximately 1 pH unit on either side of the
first pKa of 2.34 and the second pKa of 9.60.
• Another important piece of information derived from the titration curve of an
amino acid is the relationship between its net charge and the pH of the solution.
At pH 5.97, the point of inflection between the two stages in its titration curve,
glycine is present predominantly as its dipolar form, fully ionized but with no net
electric charge. The characteristic pH at which the net electric charge is zero is
called the isoelectric point or isoelectric pH, designated pI.
Glycine has a net negative charge at any pH above its pI and will thus
move toward the positive electrode (the anode) when placed in an
electric field. At any pH below its pI, glycine has a net positive charge
and will move toward the negative electrode (the cathode)
Resources: http://faculty.une.edu/com/courses/bionut/distbio/obj-512/Chap6-charge%20on%20protein.html
Proteins Page 4
Proteins Page 5
A scale combining hydrophobicity and hydrophilicity of R groups. The values reflect the free energy
(DG) of transfer of the amino acid side chain from a hydrophobic solvent to water. This transfer is
favorable (DG , 0; negative value in the index) for charged or polar amino acid side chains, and
unfavorable (DG . 0; positive value in the index) for amino acids with nonpolar or more hydrophobic side
chains.
Proteins Page 6
Peptides and Proteins
Thursday, 23 September, 2021 04:25 PM
Peptides Are Chains of Amino Acids
• Two amino acid molecules can be covalently joined through a substituted amide linkage, termed a peptide bond, to yield a dipeptide.
• Such a linkage is formed by removal of the elements of water (dehydration) from the -carboxyl group of one amino acid and the -amino group
of another. Peptide bond formation is an example of a condensation reaction, a common class of reactions in living cells.
• An amino acid unit in a peptide is often called a residue (the part left over after losing the elements of water—a hydrogen atom from its
amino group and the hydroxyl moiety from its carboxyl group). In a peptide, the amino acid residue at the end with a free -amino group is the
amino-terminal (or N-terminal) residue; the residue at the other end, which has a free carboxyl group, is the carboxylterminal (C-terminal)
residue. When an amino acid sequence of a peptide, polypeptide, or protein is displayed, the aminoterminal end is placed on the left, the
carboxyl-terminal end on the right. The sequence is read left to right, beginning with the amino-terminal end.
Peptides Can Be Distinguished by Their Ionization Behavior

• The acid-base behavior of a peptide can be predicted from its free -amino and -carboxyl groups combined with the nature and number of its
ionizable R groups
• Like free amino acids, peptides have characteristic titration curves and a characteristic isoelectric pH (pI) at which they do not move in an
electric field.
Biologically Active Peptides and Polypeptides Occur in a Vast Range of Sizes and Compositions
• Proteins can be very long polypeptide chains of 100 to several thousand amino acid residues. However, some naturally occurring peptides have
only a few amino acid residues.
• Some proteins are composed of several noncovalently associated polypeptide chains, called subunits.
• A few proteins contain two or more polypeptide chains linked covalently. For example, the two polypeptide chains of insulin are linked by
disulfide bonds. In such cases, the individual polypeptides are not considered subunits but are commonly referred to simply as chains.
• The amino acid composition of proteins is also highly variable. The 20 common amino acids almost never occur in equal amounts in a protein.
Some amino acids may occur only once or not at all in a given type of protein; others may occur in large numbers.
• We can calculate the approximate number of amino acid residues in a simple protein containing no other chemical constituents by
dividing its molecular weight by 110. Although the average molecular weight of the 20 common amino acids is about 138, the smaller amino
acids predominate in most proteins. If we take into account the proportions in which the various amino acids occur in an average protein (the
averages are determined by surveying the amino acid
compositions of more than 1,000 different proteins), the average molecular weight of protein amino acids is nearer to 128. Because a molecule
of water (Mr 18) is removed to create each peptide bond, the average molecular weight of an amino acid residue in a protein is about 128 - 18 =
110.
Some Proteins Contain Chemical Groups Other Than Amino Acids
• Simple proteins yield only amino acids on hydrolysis; conjugated proteins contain in addition some other component, such as a metal or organic
prosthetic group
Proteins Page 7
Determining Protein 1º Structure
Sunday, 26 September, 2021 01:25 AM
Differences in protein function result from differences in amino acid composition and sequence. Some variations in sequence m ay
occur in a particular protein, with little or no effect on its function.
The structure of large molecules such as proteins can be described at several levels of complexity, arranged in a kind of con ceptual
hierarchy. Four levels of protein structure are commonly defined.
A description of all covalent bonds (mainly peptide bonds and disulfide bonds) linking amino acid residues in a polypeptide chain
is its primary structure. The most important element of primary structure is the sequence of amino acid residues. Secondary
structure refers to particularly stable arrangements of amino acid residues giving rise to recurring structural patterns. Tertiary
structure describes all aspects of the three-dimensional folding of a polypeptide. When a protein has two or more polypeptide
subunits, their arrangement in space is referred to as quaternary
structure.
Amino Acid Sequencing - Edman Degradation

The Edman degradation procedure labels and removes only the amino-terminal residue from a peptide, leaving all other peptide
bonds intact. The labelled amino acid is identified using HPLC. The process is repeated until, typically, as many as 40 seque ntial
amino acid residues are identified. Therefore, this works only for small polypeptides.
Proteins Page 8
To determine the sequence of large proteins, we need to do two things --
(i) eliminate disulfide binds
(ii) cleave proteins precisely into smaller polypeptides.
Enzymes called proteases catalyze the hydrolytic cleavage of peptide bonds. Some proteases cleave only the peptide bond adjacent
to particular amino acid residues and thus fragment a polypeptide chain in a predictable and reproducible way.
In classical sequencing, a large protein would be cleaved into fragments twice, using a different protease or cleavage
reagent each time so that the fragment endpoints would be different. Both sets of fragments would be purified and
Proteins Page 9
reagent each time so that the fragment endpoints would be different. Both sets of fragments would be purified and
sequenced. The order in which the fragments appeared in the original protein could then be determined by examining the
overlaps in sequence between the two sets of fragments.
Amino Acid Sequencing - Mass Spectrometry
Mass spectroscopy is an analytic technique by which chemical substances are identified by the sorting of gaseous ions in electric
and magnetic fields according to their mass-to-charge ratios. In order to measure the characteristics of individual molecules, a mass
spectrometer converts molecules to ions so that they can be moved about and manipulated by external electric and magnetic
fields. The three essential functions of a mass spectrometer, and the associated components, are:
1. A small sample is ionized, usually to cations by loss of an electron. The Ion Source
2. The ions are sorted and separated according to their mass and charge. The Mass Analyzer
3. The separated ions are then measured, and the results displayed on a chart. The Detector
▫ Because ions are very reactive and short-lived, their formation and manipulation must be conducted in a vacuum.
Atmospheric pressure is around 760 torr (mm of mercury). The pressure under which ions may be handled is roughly 10-5 to
10-8 torr (less than a billionth of an atmosphere).
▫ The heart of the spectrometer is the ion source. Here molecules of the sample (black dots) are bombarded by electrons (light
blue lines) issuing from a heated filament. This is called an EI (electron-impact) source. Gases and volatile liquid samples are
allowed to leak into the ion source from a reservoir (as shown). Non-volatile solids and liquids may be introduced directly.
Cations formed by the electron bombardment (red dots) are pushed away by a charged repeller plate (anions are attracted to
it), and accelerated toward other electrodes, having slits through which the ions pass as a beam. Some of these ions fragment
into smaller cations and neutral fragments. A perpendicular magnetic field deflects the ion beam in an arc whose radius is
proportional to the mass of each ion. Lighter ions are deflected more than heavier ions. By varying the strength of the
magnetic field, ions of different mass can be focused progressively on a detector fixed at the end of a curved tube (also under
a high vacuum).
▫ When a high energy electron collides with a molecule it often ionizes it by knocking away one of the molecular electrons
(either bonding or non-bonding). This leaves behind a molecular ion (colored red in the following diagram). Residual energy
from the collision may cause the molecular ion to fragment into neutral pieces (colored green) and smaller fragment ions
(colored pink and orange). The molecular ion is a radical cation, but the fragment ions may either be radical cations (pink) or
carbocations (orange), depending on the nature of the neutral fragment.
Proteins Page 10
▫
▫ fsf
Proteins Page 11
Overview of Proteins Structure
Proteins Page 12
Secondary Structure of Proteins
Proteins Page 13
Tertiary & Quaternary Structure of Proteins
Proteins Page 14
Protein Denaturation & Folding
Proteins Page 15
Protein Functions
Tuesday, 14 September, 2021 04:26 PM
What Biological Functions Do Proteins Perform?

Proteins fulfill the following biochemical roles:
1. Enzyme catalysis. Enzymes are protein catalysts, capable of enhancing the rates of reactions by
factors of
up to 1014. For example, the enzyme carbonic anhydrase catalyzes the reaction CO2 + 2O H+ +
HCO3−   ⧫  7 over the uncatalyzed reaction.
2. Transport and storage. Many small molecules are transported both inside and outside cells bound
to
carrier proteins. Examples include the oxygen transport and storage proteins hemoglobin and
myoglobin,
respectively. The proteins in membranes often permit the passage of both small and large molecules
through membranes.
3. Mechanical structure and support. Collagen provides the tensile strength in skin, teeth, and bone;
keratin
is the major component of hair and fur; and fibroin is one of two proteins that comprise spider silk.
4. Motion. Muscle contraction is accomplished by the interaction of filaments composed of two
different
proteins, actin and myosin. Kinesin moves protein cargoes around cells along microtubule “rails”
formed
by tubulin.
5. Recognition. Antibodies are proteins that form part of the immune response in mammals in the
presence
of foreign invaders. Other proteins form the major histocompatibility complex (MHC), which presents
small peptides from viruses and other invaders for recognition by the immune system.
6. Information and control. Stimuli external to a cell, such as hormone signals or light, are detected by
specific proteins, receptors and the photosystem, for example, that transfer signals to the cell interior.
The visual protein rhodopsin is located in the membranes of retinal cells. Other proteins control the
basic
Structure of Proteins
cellular functions of transcription and translation.
7. Exotic functions. Fish living in the very cold waters in the Antarctic Ocean require antifreeze
proteins to structure of proteins =
• Primary
prevent ice formation in their cells.
Linear Sequence of amino acids
• 1º structure is produced by ribosome
in cytosol or ER
• This linear sequence then folds into
the protein's native 3D structure
Proteins Page 16
Enzymes
Proteins Page 17
Amino Acid Oxidation & Production of Urea
Proteins Page 18
Biosynthesis of Amino Acids
Proteins Page 19
Protein Metabolism
Proteins Page 20
Working with Proteins
Friday, 15 October, 2021 06:55 PM
wou.edu
Chapter 3: Investigating Proteins – Chemistry

124-157 minutes
3.1 Protein Purification

3.2 Protein Identification and Visualization
3.3 Protein Synthesis and Sequencing
3.4 Protein Structure Elucidation
3.5 Proteome Analysis
3.6 References
Protein purification is a series of processes intended to isolate one or a few proteins from a complex mixture, usually cells, tissues or
whole organisms. Protein purification is vital for the characterization of the function, structure and interactions of the pr otein of
interest. The purification process may separate the protein and non-protein parts of the mixture, and finally separate the desired
protein from all other proteins. Separation of one protein from all others is typically the most laborious aspect of protein purification.
Separation steps usually exploit differences in protein size, physico-chemical properties, binding affinity and biological activity. The
pure result may be termed protein isolate.
Protein purification is either preparative or analytical.

Preparative purifications aim to produce a relatively large quantity of Analytical purification produces a relatively small
purified proteins for subsequent use. Examples include the preparation of amount of a protein for a variety of research or
commercial products such as enzymes (e.g. lactase), nutritional proteins analytical purposes, including identification,
(e.g. soy protein isolate), and certain biopharmaceuticals (e.g. insulin). quantification, and studies of the protein’s structure,
Several preparative purifications steps are often deployed to remove bi- post-translational modifications and function. Pepsin and
products, such as host cell proteins, which poses as a potential threat to urease were the first proteins purified to the point that
the patient’s health. they could be crystallized.
Extraction
If the protein of interest is not secreted by the organism into the surrounding solution, the first step of each purification process is
the disruption of the cells containing the protein. Depending on how fragile the protein is and how stable the cells are, one could, for
instance, use one of the following methods:
i) repeated freezing and thawing,
ii) sonication,
iii) homogenization by high pressure (French press),
iv) homogenization by grinding (bead mill), and
v) permeabilization by detergents (e.g. Triton X-100) and/or enzymes (e.g. lysozyme).
Finally, the cell debris can be removed by centrifugation so that the proteins and other soluble compounds remain in the supe rnatant.
Also proteases are released during cell lysis, which will start digesting the proteins in the solution. If the protein of int erest is
sensitive to proteolysis, it is recommended to proceed quickly, and to keep the extract cooled, to slow down the digestion.
Alternatively, one or more protease inhibitors can be added to the lysis buffer immediately before cell disruption. Sometimes it is also
necessary to add DNAse in order to reduce the viscosity of the cell lysate caused by a high DNA content.
Precipitation and Differential Solubilization
In bulk protein purification, a common first step to isolate proteins is precipitation using a salt such as ammonium sulfate (NH4)2SO4.
This process is called Salting In or Salting Out (Figure 3.1) This is performed by adding increasing amounts of ammonium sulfate and
collecting the different fractions of precipitate protein. Ammonium sulfate is often used as it is highly soluble in water, h as relative
freedom from temperature effects and typically is not harmful to most proteins. Furthermore, ammonium sulfate can be removed by
dialysis (Figure 3.2). The hydrophobic groups on the proteins get exposed to the atmosphere, attract other protein hydrophobic
groups and get aggregated. Protein precipitated will be large enough to be visible. One advantage of this method is that it can be
performed inexpensively with very large volumes.
Proteins Page 21
Figure 3.1 Salting In and Salting Out. During the salting in process, salt molecules increase the solubility of proteins by reducing
the electrostatic interactions between protein molecules. As the salt concentration is increased, protein-protein interactions become
more energetically favorable than protein-solvent interactions and the proteins precipitate from solution.
Image derived fromMichel Awkal
The first proteins to be purified are water-soluble proteins. Purification of integral membrane proteins requires disruption of the cell
membrane in order to isolate any one particular protein from others that are in the same membrane compartment. Sometimes a
particular membrane fraction can be isolated first, such as isolating mitochondria from cells before purifying a protein loca ted in a
mitochondrial membrane. A detergent such as sodium dodecyl sulfate (SDS) can be used to dissolve cell membranes and keep
membrane proteins in solution during purification; however, because SDS causes denaturation, milder detergents such as Triton X-100
or CHAPS can be used to retain the protein’s native conformation during complete purification.
Figure 3.2 Dialysis. The process of dialysis separates dissolved molecules by their size. The biological sample is placed inside a closed
membrane, where the protein of interest is too large to pass through the pores of the membrane, but through which smaller ion s can
easily pass. As the solution comes to equilibrium, the ions become evenly distributed throughout the entire solution, while t he protein
remains concentrated in the membrane. This reduces the overall salt concentration of the suspension.
Image adapted fromGjk003
Ultracentrifugation
Centrifugation is a process that uses centrifugal force to separate mixtures of particles of varying masses or densities suspended in a
liquid. When a vessel (typically a tube or bottle) containing a mixture of proteins or other particulate matter, such as bact erial cells, is
rotated at high speeds, the inertia of each particle yields a force in the direction of the particles velocity that is propor tional to its
mass. The tendency of a given particle to move through the liquid because of this force is offset by the resistance the liqui d exerts on
the particle. The net effect of “spinning” the sample in a centrifuge is that massive, small, and dense particles move outwar d faster
than less massive particles or particles with more “drag” in the liquid. When suspensions of particles are “spun” in a centri fuge, a
“pellet” may form at the bottom of the vessel that is enriched for the most massive particles with low drag in the liquid.
Non-compacted particles remain mostly in the liquid called “supernatant” and can be removed from the vessel thereby separating th e
Proteins Page 22
Non-compacted particles remain mostly in the liquid called “supernatant” and can be removed from the vessel thereby separating th e
supernatant from the pellet. The rate of centrifugation is determined by the angular acceleration applied to the sample, typi cally
measured in comparison to the g. If samples are centrifuged long enough, the particles in the vessel will reach equilibrium wherein the
particles accumulate specifically at a point in the vessel where their buoyant density is balanced with centrifugal force. Su ch an
“equilibrium” centrifugation can allow extensive purification of a given particle.
In sucrose gradient centrifugation, a linear concentration gradient of sugar (typically sucrose, glycerol, or a silica based density
gradient media, like Percoll) is generated in a tube such that the highest concentration is on the bottom and lowest on top. Percoll is a
trademark owned by GE Healthcare companies. A protein sample is then layered on top of the gradient and spun at high speeds i n an
ultracentrifuge. This causes heavy macromolecules to migrate towards the bottom of the tube faster than lighter material. Dur ing
centrifugation in the absence of sucrose, as particles move farther and farther from the center of rotation, they experience more and
more centrifugal force (the further they move, the faster they move). The problem with this is that the useful separation ran ge of
within the vessel is restricted to a small observable window. A properly designed sucrose gradient will counteract the increa sing
centrifugal force so the particles move in close proportion to the time they have been in the centrifugal field. Samples sepa rated by
these gradients are referred to as “rate zonal” centrifugations. After separating the protein/particles, the gradient is then
fractionated and collected.
Figure 3.3 Sucrose Density Gradient.

back to the top
Purification Strategy
Choice of a starting material is key to the design of a purification process. In a plant or animal, a particular protein usua lly isn’t
distributed homogeneously throughout the body; different organs or tissues have higher or lower concentrations of the protein . Use
of only the tissues or organs with the highest concentration decreases the volumes needed to produce a given amount of purifi ed
protein. If the protein is present in low abundance, or if it has a high value, scientists may use recombinant DNA technology to develop
cells that will produce large quantities of the desired protein (this is known as an expression system). Recombinant expressi on allows
the protein to be tagged, e.g. by a His-tag or Strep-tag to facilitate purification, reducing the number of purification steps required.
These techniques will be discussed in greater detail in Chapter 5.
An analytical purification generally utilizes three properties to separate proteins. First, proteins may be purified accordin g to their
isoelectric points by running them through a pH graded gel or an ion exchange column. Second, proteins can be separated accor ding to
their size or molecular weight via size exclusion chromatography or by SDS -PAGE (sodium dodecyl sulfate-polyacrylamide gel
electrophoresis) analysis. Proteins are often purified by using 2D-PAGE and are then analysed by peptide mass fingerprinting to
establish the protein identity. This is very useful for scientific purposes and the detection limits for protein are nowadays very low
and nanogram amounts of protein are sufficient for their analysis. Thirdly, proteins may be separated by polarity/hydrophobic ity via
high performance liquid chromatography or reversed-phase chromatography. Gel electrophoresis techniques are discussed in more
detail in Section 3.2. This section will focus predominantly on chromatographic separations.
For preparative protein purification, the purification protocol generally contains one or more chromatographic steps. The bas ic
procedure in chromatography is to flow the solution containing the protein through a column packed with various materials. Di fferent
proteins interact differently with the column material, and can thus be separated by the time required to pass the column, or the
conditions required to elute the protein from the column. Usually proteins are detected as they are coming off the column by their
absorbance at 280 nm. Many different chromatographic methods exist, with the most common described below:
Size Exclusion Chromatography (also known as Gel Filtration Chromatography)
Chromatography can be used to separate protein in solution or under denaturing conditions by using porous gels. This techniqu e is
known as size exclusion chromatography. The principle is that smaller molecules have to traverse a larger volume in a porous matrix.
Consequentially, proteins of a certain range in size will require a variable volume of eluent (solvent) before being collecte d at the other
end of the column of gel. Thus, proteins will be separated based on their size (Figure 3.4).
In the context of protein purification, the eluent is usually pooled in different test tubes. All test tubes containing no me asurable
trace of the protein to purify are discarded. The remaining solution is thus made of the protein to purify and any other simi larly-sized
proteins.
Proteins Page 23
Figure 3.4 Size Exclusion Chromatography. Also known as Gel Filtration Chromatography, is a low resolution isolation method that
involves the use of beads that have tiny “tunnels” in them that each have a precise size. The size is referred to as an “excl usion limit,”
which means that molecules above a certain molecular weight will not fit into the tunnels. Molecules with sizes larger than t he
exclusion limit do not enter the tunnels and pass through the column relatively quickly by making their way between the beads . Smaller
molecules, which can enter the tunnels, do so, and thus, have a longer path that they take in passing through the column. Bec ause of
this, molecules larger than the exclusion limit will leave the column earlier, while smaller molecules that pass through the beads will
elute from the column later. This method allows separation of molecules by their size.
Image fromDr. Kevin Ahern and Indira Rajagopal
Hydrophobic Interaction Chromatography (HIC)

HIC media is amphiphilic, with both hydrophobic and hydrophilic regions, allowing for separation of proteins based on their s urface
hydrophobicity. Target proteins and their product aggregate species tend to have different hydrophobic properties and removin g
them via HIC further purifies the protein of interest. Additionally, the environment used typically employs less harsh denatu ring
conditions than other chromatography techniques, thus helping to preserve the protein of interest in its native and functiona l state. In
pure water, the interactions between the resin and the hydrophobic regions of protein would be very weak, but this interactio n is
enhanced by applying a protein sample to HIC resin in high ionic strength buffer. The ionic strength of the buffer is then re duced to
elute proteins in order of decreasing hydrophobicity (Figure 3.5).
Figure 3.5 Hydrophobic Interaction Chromatography. The column matrix, shown in blue has a hydrophobic ligand covalently attached.
In high salt conditions, proteins will bind to the matrix with differing affinity, with more hydrophobic proteins (shown in y ellow)
binding more tightly than more hydrophilic proteins (shown in green) When the salt concentration is decreased, proteins that are more
hydrophilic will be released first, followed more hydrophobic proteins.
Ion Exchange Chromatography

Ion exchange chromatography separates compounds according to the nature and degree of their ionic charge. The column to be us ed is
selected according to its type and strength of charge. Anion exchange resins have a positive charge and are used to retain an d
separate negatively charged compounds (anions), while cation exchange resins have a negative charge and are used to separate
Proteins Page 24
separate negatively charged compounds (anions), while cation exchange resins have a negative charge and are used to separate
positively charged molecules (cations).
Before the separation begins a buffer is pumped through the column to equilibrate the opposing charged ions. Upon injection o f the
sample, solute molecules will exchange with the buffer ions as each competes for the binding sites on the resin. The length o f
retention for each solute depends upon the strength of its charge. The most weakly charged compounds will elute first, follow ed by
those with successively stronger charges. Because of the nature of the separating mechanism, pH, buffer type, buffer concentr ation,
and temperature all play important roles in controlling the separation.
Figure 3.6 demonstrates a type of ion exchange column known as a cation exchange column. In this case, the support consists of tiny
beads to which are attached chemicals possessing a charge. Each charged molecule has a counter -ion. The figure shows the beads
(blue) with negatively charged groups (red) attached. In this example, the counter -ion is sodium, which is positively charged. The
negatively charged groups are unable to leave the beads, due to their covalent attachment, but the counter - ions can be “exchanged”
for molecules of the same charge. Thus, a cation exchange column will have positively charged counter-ions and positively charged
compounds present in a mixture passed through the column will exchange with the counter -ions and “stick” to the negatively charged
groups on the beads. Molecules in the sample that are neutral or negatively charged will pass quickly through the column. On the other
hand, in anion exchange chromatography, the chemical groups attached to the beads are positively charged and the counter -ions are
negatively charged. Molecules in the sample that are negatively charged will “stick” and other molecules will pass through qu ickly. To
remove the molecules “stuck” to a column, one simply needs to add a high concentration of the appropriate counter -ions to displace and
release them. This method allows the recovery of all components of the mixture that share the same charge.
Ion exchange chromatography is a very powerful tool for use in protein purification and is frequently used in both analytical and
preparative separations.
Figure 3.6 Cation Exchange Chromatography. In this diagram the negatively charged molecules (shown in red) are covalently
attached to the column matrix beads (shown in blue). Sodium ions (Na+) are the counter ions that are replaced by positively c harged
proteins within the protein mixture. Neutral and negatively charged proteins do not stick and will pass through the column. T he
positively charged proteins can then be eluted from the column by adding higher concentrations of the counter ion (in this ca se the
sodium ions).
Image fromKevin Ahern and Indira Rajagopal
Affinity Chromatography is a separation technique based upon molecular conformation, which frequently utilizes application sp ecific
resins. These resins have ligands (small molecules) attached to their surfaces which are specific for and will bind with the compounds
to be separated. Most frequently, these ligands function in a fashion similar to that of antibody -antigen interactions. This “lock and
key” fit between the ligand and its target compound makes it highly specific, frequently generating a single peak, while all else in the
sample is unretained (Figure 3.7).
For example, many membrane proteins are glycoproteins and can be purified by lectin affinity chromatography. Detergent -solubilized
proteins can be allowed to bind to a chromatography resin that has been modified to have a covalently attached lectin. Protei ns that do
not bind to the lectin are washed away and then specifically bound glycoproteins can be eluted by adding a high concentration of a
sugar that competes with the bound glycoproteins at the lectin binding site. Some lectins have high affinity binding to oligo saccharides
of glycoproteins that is hard to compete with sugars, and bound glycoproteins need to be released by denaturing the lectin.
Proteins Page 25
Figure 3.7 Example of Affinity Chromatography. In this example, protein P1 has affinity for ligand Z and will bind to the column
while proteins P2 and P3 will pass through the column. Protein P1 can then be eluted from the column using high concentrations of free
ligand Z.
A common technique involves engineering a sequence of 6 to 8 histidine residues into the N - or C-terminal of a recombinant protein.
The polyhistidine binds strongly to divalent metal ions such as nickel and cobalt. The protein can be passed through a column containing
immobilized nickel ions, which binds the polyhistidine tag. All untagged proteins pass through the column. The protein can be eluted
with imidazole, which competes with the polyhistidine tag for binding to the column, or by a decrease in pH (typically to 4.5 ), which
decreases the affinity of the tag for the resin. While this procedure is generally used for the purification of recombinant p roteins
with an engineered affinity tag (such as a 6xHis tag), it can also be used for natural proteins with an inherent affinity for divalent
cations.
Immunoaffinity chromatography
A special type of affinity chromatography is Immunoaffinity chromatography (Figure 3.8). This technique uses the specific bin ding of
an antibody with its antigen (target molecule that the antibody will bind with selectively) to purify the protein of interest . The
procedure involves immobilizing an antibody to a solid substrate (e.g. a porous bead or a membrane), which then selectively b inds the
target, while everything else flows through. The target protein can be eluted by changing the pH or the salinity. The immobil ized
ligand can be an antibody (such as Immunoglobulin G) or it can be a protein (such as Protein A). Because this method does not involve
engineering in a tag, it can be used for proteins from natural sources. Antibody structure and their use in protein identific ation will be
discussed in greater detail in Section 3.2.
Proteins Page 26
Figure 3.8. An Antigen Immunoprecipitation Experiment. The antibody is either pre-immobilized to a solid support (left) or
immobilized using antibody binding proteins after incubation with the sample (right). Immobilization allows the immune comple x to be
extracted from the complex sample, washed and eluted providing a high enrichment of the protein under investigation
Image fromThe Human Atlas Project
back to the top
High Performance Liquid Chromatography (HPLC) and Fast Protein Liquid Chromatography (FPLC)
High performance liquid chromatography or high pressure liquid chromatography (HPLC) is a form of chromatography applying high
pressure to drive the solutes through the column faster. This means that the diffusion is limited and the resolution is impro ved. The
most common form is “reversed phase” HPLC, where the column material is hydrophobic. The proteins are eluted by a gradient of
water and increasing amounts of an organic solvent, such as acetonitrile. The proteins elute according to their hydrophobicit y. After
purification by HPLC the protein is in a solution that only contains volatile compounds, and can easily be lyophilized (freeze dried).
HPLC purification frequently results in denaturation of the purified proteins and is thus not applicable to proteins that do not
spontaneously refold.
Due to the drawbacks of HPLC, an alternative technique using a lower pressure system was developed and is called Fast protein liquid
chromatography (FPLC). FPLC is a form of liquid chromatography that is often used to analyze or purify mixtures of proteins. As in
other forms of chromatography, separation is possible because the different components of a mixture have different affinities for
two materials, a moving fluid (the “mobile phase”) and a porous solid (the stationary phase). In FPLC the mobile phase is an aqueous
solution, or “buffer”. The buffer flow rate is controlled by a positive-displacement pump and is normally kept constant, while the
composition of the buffer can be varied by drawing fluids in different proportions from two or more external reservoirs. The
stationary phase is a resin composed of beads, usually of cross-linked agarose, packed into a cylindrical glass or plastic column. FPLC
resins are available in a wide range of bead sizes and surface ligands depending on the application.
In the most common FPLC strategy, an ion exchange resin is typically chosen (Figure 3.9). A mixture containing one or more proteins
of interest is dissolved in 100% buffer A and pumped into the column. The proteins of interest bind to the resin while other
components are carried out in the buffer. The total flow rate of the buffer is kept constant; however, the proportion of Buff er B (the
“elution” buffer) is gradually increased from 0% to 100% according to a programmed change in concentration (the “gradient”). Buffer B
contains high concentrations of the exchanger ion. Thus as the concentration of the Buffer B gradually increases, bound prote ins will
dissociate depending on their ionic interactions with the column matrix and appear in the eluant. The eluant passes through t wo
detectors which measure salt concentration (by conductivity) and protein concentration (by absorption of ultraviolet light at a
wavelength of 280nm). As each protein is eluted it appears in the eluant as a “peak” in protein concentration and can be coll ected for
further use.
FPLC was developed and marketed in Sweden by Pharmacia in 1982 and was originally called fast performance liquid chromatography
Proteins Page 27
FPLC was developed and marketed in Sweden by Pharmacia in 1982 and was originally called fast performance liquid chromatography
to contrast it with HPLC or high-performance liquid chromatography. FPLC is generally applied only to proteins; however, because of
the wide choice of resins and buffers it has broad applications. In contrast to HPLC the buffer pressure used is relatively l ow,
typically less than 5 bar, but the flow rate is relatively high, typically 1-5 ml/min. FPLC can be readily scaled from analysis of
milligrams of mixtures in columns with a total volume of 5 ml or less to industrial production of kilograms of purified prote in in columns
with volumes of many liters.
Figure 3.9 Typical FPLC System. A. Scheme of basic compounents and typical flow path for a chromatography system. B. Picrue of GE
Healthcare AKTA FPLC apparatus.
Image provided byLaVerde, V., Dominici, P. and Astegno, A. (2017) Bio-protocol 7(8): e2230.
Purification Scheme
During the protein purification process it is necessary to have a quantitative system to determine how much protein has been purified,
what concentration the protein represents from the original mixture, how biologically active the purified protein is, and the overall
purity of the protein. This will help guide and optimize the purification method being developed. Ineffective separation techniques can
be disregarded and other techniques that give higher yield or that retain biologically activity of the protein can be adopted .
Thus, each step in the purification scheme is quantitatively evaluated for the following parameters: total protein, total act ivity,
specific activity, yield, purification level. Each of these parameters will be defined within the sample protocol given below .
Pretend you are a researcher that wants to isolate a novel, unknown protein from a bacterial culture. You grow 500 ml of the bacteria
overnight at 37oC and harvest the bacteria by centrifugation. You remove the culture broth and retain the bacterial pellet. You then
lyse the bacteria using freeze/thaw in 10 mL of reaction buffer. You then centrifuge the lysed bacteria to remove the insolub le
materials and retain the supernatent that contains the soluble proteins. Your protein of interest has a biological activity t hat you can
measure using a simple assay that causes a color change in the reaction mixture (Figure 3.10). You also note that this reacti on rate
increases with increasing concentrations of your protein supernatent (Figure 3.10)
Figure 3.10. Example of a Chemical Reaction that causes a color change from orange to brown depending on increasing
concentration.
Image from: Ludwig, N., et. al. (2015) on Research Gate
At this point, you can measure your baseline concentrations for the first purification level (bacterial lysis and removal of insoluble
proteins and other cellular debris by centrifugation).
Total Protein is calculated by measuring the concentration in a fraction of your sample, and then multiplying that by the total volume
of your sample. In this case, you are starting with 10 mL of supernatent. In a typical assay to measure protein concentration , you will
use 50 – 200 μL of sample to determine the protein concentration. For example, if you calculate that there is 7.5 μg/μL in your initia l
assay, you would need to convert that value into mg/mL and then multiply it by 10 mL for a total of 75 mg of protein in 10 mL of
supernatant (Table 3.1)
Total Activity is measured as the enzyme activity within the assay, multiplied by the total volume of the sample. For example, in the
initial sample, you might use 5 to 50 μL of sample in your biological reaction (Figure 3.10). If you calculated the activity in your assay to
be 2.5 units/μL, this would be equivalent to 2,500 units/mL or 25,000 units/10 mL of supernatant. Note that, the enzyme unit, or
international unit for enzyme (symbol U, sometimes also IU) is a unit of enzyme’s catalytic activity. 1 U (μmol/min) is defined as the
amount of the enzyme that catalyzes the conversion of one micromole of substrate per minute under the specified conditions of the
assay method.
Specific Activity is measured by dividing the Total Activity by the Total Protein. In our example, 25,000 units divided by 75 mg of
protein = 333.3 units/mg.
Yield is a measure of the biological activity retained in the sample after each purification step. The amount in the first step is set to
Proteins Page 28
Yield is a measure of the biological activity retained in the sample after each purification step. The amount in the first step is set to
be 100%. All subsequent yield steps will be evaluated using the first purification step. It is calculated by dividing the tot al activity of
the current step, by the total activity of the first step and then multiplying by 100.
Purification level evaluates the purity of the protein of interest by dividing the specific activity calculated after each purification
step by the specific activity of the first purification step. Thus, the first step always has a value of 1.
Table 3.1 Typical Protein Purification Scheme
Note that after each purification step that the Total Protein goes down, as you are purifying your protein away from other proteins in
the mixture. Total Activity also goes down with each purification step, as some of your protein of interest is also lost at each
purification step, because (1) some protein will stick to the test tubes and glassware, (2) some protein won’t bind with 100% efficiency
to your column matrix, (3) some protein may bind too tightly to be removed from the column matrix during elution, and (4) some
protein may be denatured or degraded during the purification process.
The amount of your protein of interest that is lost is represented within the overall percent yield for each purification step. If the
percent yield is too low alternative purification methods should be explored.
Note that in a good protein purification scheme that the specific activity should go up substantially with each level of purification as
the amount of your protein of interest makes up a greater percentage of the total protein within that fraction. If the specif ic activity
only increases modestly within a purification step, or if it decreases during a purification step, this could indicate that ( 1) your protein
of interest is being substantially lost at that step, (2) that your protein of interest is being denatured or degraded and is no longer
biologically active, or (3) that a required cofactor or binding protein is being reduced at that purification step. Additiona l experiments
may need to be conducted to determine which of the causes predominates, so that steps can be taken to reduce protein inactiva tion.
For example, many proteins are temperature sensitive and will degrade or denature at room temperature. Completing purification
steps on ice can often reduce degradation.
Overall, the fold increase in purification level should increase exponentially during the purification process. Note that in our example,
if after 4 steps of purification our proteins is close to 95% pure, this would indicate that our protein of interest makes up
approximately 1.24% of the total protein within the sample.
back to the top

Analytical techniques that can be used to positively identify or visualize a protein of interest within a mixture can also be a valuable
tool to understanding the biological activity and significance of a protein within a living system and can also be used to he lp guide
protein purification schemes.
Gel Electrophoresis
(work derived fromMagdeldin, S.)
Agarose is a natural linear polymer extracted from seaweed that forms a gel matrix by hydrogen -bonding when heated in a buffer and
allowed to cool. For most applications, only a single-component agarose is needed and no polymerization catalysts are required (Figure
3.11). Therefore, agarose gels are simple and rapid to prepare. They are the most popular medium for the separation of modera te and
large-sized nucleic acids and have a wide range of separation but a relatively low resolving power, since the bands formed in the g els
tend to be fuzzy and spread apart. This is a result of pore size and cannot be largely controlled. These and other advantages and
disadvantages of using agarose gels for electrophoresis are summarized in Table 3.2. Agarose gels are not typically used for protein
samples and won’t be discussed in this chapter further. However, they will be revisted in Chapter 5 covering nucleic acid techniques.
Table 3.2. Advantages and Disadvantages of Agarose Gel Electrophoresis.
Polyacrylamide gels are chemically cross-linked gels formed by the polymerization of acrylamide with a cross-linking agent, usually
N,N’-methylenebisacrylamide (Figure 3.11). The reaction is a free radical polymerization, usually carried out with ammonium persul fate
as the initiator and N,N,N’,N’-tetramethylethylendiamine (TEMED) as the catalyst. Although the gels are generally more difficult to
prepare and handle, involving a longer time for preparation than agarose gels, they have major advantages over agarose gels. They have
a greater resolving power, can accommodate larger quantities of sample without significant loss in resolution and the purity of the
Proteins Page 29
a greater resolving power, can accommodate larger quantities of sample without significant loss in resolution and the purity of the
sample recovered from polyacrylamide gels is extremely high. Moreover, the pore size of the polyacrylamide gels can be altere d in an
easy and controllable fashion by changing the concentrations of the two monomers. Thus, it is commonly used to separate prote ins and
smaller fragments of DNA. It should be noted that polyacrylamide is a neurotoxin (when unpolymerized), but with proper labora tory
care it is no more dangerous than various commonly used chemicals. Some advantages and disadvantages of using polyacrylamide gels
for electrophoresis are depicted in Table 3.3.
Table 3.3. Advantages and Disadvantages of Polyacrylamide Gel Electrophoresis.
Hydrated gel networks have many desirable properties for electrophoresis. They allow a wide variety of mechanically stable
experimental formats such as horizontal/vertical electrophoresis in slab gels or electrophoresis in tubes or capillaries. The mechanical
stability also facilitates post electrophoretic manipulation making further experimentation possible such as blotting, electr o-elution or
mass spectral identification /finger printing of intact proteins or of proteins digested in gel slices. Since gels used in bi ochemistry are
chemically rather unreactive, they interact minimally with biomolecules during electrophoresis allowing separation based on p hysical
rather than chemical differences between sample components.
Figure 3.11 Gels Commonly Used in Electrophoresis. (A) Agarose is composed of agarbiose, (B) The polymerization of acrylamide and
bisacrylamide to form polyacrylamide gel. The polymerization reaction is initiated by persulfate radicals and catalyzed by TE MED.
Image fromMagdeldin, S.
Gel electrophoresis of proteins with a polyacrylamide matrix, commonly called polyacrylamide gel electrophoresis (PAGE) is
undoubtedly one of the most widely used techniques to characterize complex protein mixtures. It is a convenient, fast and ine xpensive
method because they require only the order of micrograms quantities of protein. They are usually run in a vertical format and the gel
rigs contain an upper and lower buffer reservoir (Fig. 3.12A). The samples are loaded in wells that contact the upper buffer reservoir
which will house the negative cathode. The proteins migrate towards the positive anode when the electric current is applied.
Note that proteins have a net electrical charge if they are in a medium having a pH different from their isoelectric point an d
therefore have the ability to move when subjected to an electric field. The migration velocity is proportional to the ratio b etween the
charges of the protein and its mass. The higher charge per unit of mass the faster the migration.
Proteins do not have a predictable structure as nucleic acids, and thus their rates of migration are not similar to each othe r.
Furthermore, they will not migrate when applying an electromotive force, when the pH of the system is the same as isoelectric point.
PAGE gels that are run in this fashion are called Native PAGE, as the proteins are still folded in their native state found in vivo. In
this situation, proteins migrate according to their charge, size and shape.
Alternatively, proteins may be denatured prior to electrophoresis. The most common way to denature the proteins is by adding a
detergent such as sodium dodecyl sulfate (SDS) (Fig 3.12B). This not only denatures the proteins, but it also coats the prote in with a
negative charge, such that all of the proteins will run towards the positive lead when placed into an electric field. This ty pe of
Proteins Page 30
negative charge, such that all of the proteins will run towards the positive lead when placed into an electric field. This ty pe of
electrophoresis is referred to as SDS-PAGE and separates proteins exclusively according to molecular weight. A reducing agent that
breaks disulfide bonds, such as dithiothreitol (DTT) is often added to the loading buffer as well, causing proteins to fully denature and
dissociate into the monomer subunits (Fig 3.12C). This ensures that the proteins migrate through the gel in direct relation t o their
size, rather than by charge or shape.
Figure 3.12 Polyacrylamide Gel Electrophoresis (PAGE). (A) Shows a typical set up for PAGE. A vertical gel is placed into a rig with
an upper and lower buffer reservoir. The upper reservoir contains the gel wells where the protein is loaded and will house th e cathode
(negative charge). The proteins will run towards the anode (positive charge) when an electric field is placed on the system, in relation
to the protein size, shape and charge. (B) Sodium dodecyl sulfate (SDS) is often used to denature proteins prior to PAGE anal ysis,
causing proteins to migrate based on size only. (C) Reducing agents, such as dithiothreitol (DTT) are often used in combinati on with
SDS, to ensure that disulfide bonds within the protein or between protein subunits are fully reduced to free cysteine residue s.
Figures from: (A) Bensaccount (B) Fdardel,and (C) Edgar181
Detection of Proteins in Gels

Proteins separated on a polyacrylamide gel can be detected by various methods, for instance dyes and silver staining (Figure 3.13).
• Dyes
The Coomassie blue staining allows detecting up to 0.2 to 0.6 µg of protein, and is quantitative (linear) up to 15 to 20 µg. It is often
used in methanol-acetic acid solutions and is discolored in isopropanol-acetic acid solutions (Fig. 1 A). For staining of 2-DE gels it is
recommended to remove ampholytes by adding trichloroacetic (TCA) to the dye and subsequently discolor with acetic acid.
• Silver staining
It is an alternative to routine staining protein gels (as well as nucleic acids and lipopolysaccharides) because its ease use and high
sensitivity (50 to 100 times more sensitive than Coomassie blue staining) (Fig. 1 B). This staining technique is particularly suitable for
two-dimensional gels.
• Detection of radioactive proteins by autoradiography
The autoradiography is a detection technique of radioactively labeled molecules that uses photographic emulsions sensitive to
radioactive particles or light produced by an intermediate molecule. The emulsion containing silver is sensitive to particula te radiation
(alpha, beta) or electromagnetic radiation (gamma, light…), so that it precipitates as metallic silver. The emulsion will dev elop as dark
precipitates in the region in which radioactive proteins are detected.
Proteins Page 31
Figure 3.13. SDS-PAGE. Proteins separated on SDS-PAGE and detected by Coomassie blue (A) and silver staining (B). Standards of
proteins to know molecular weight are also loaded at edges.
Isoelectric Focusing
This technique is based on the movement of molecules in a pH gradient. Amphoteric molecules such as amino acids and proteins are
separated in an environment where there is a difference of potential and pH gradient. The region of the anode (+) is acidic a nd the
cathode (-) is alkaline. Between them down a pH gradient such that the molecules to be separated have their isoelectric point within
the range. Substances that are initially in regions with a pH below its isoelectric point are positively charged and migrate towards the
cathode, while those that are in media with pH lower than its pI will have negative charge and migrate towards the anode (Figure
3.14). The migration will lead to a region where the pH coincide with its pI, have a zero net charge (form zwitterions) and stop. Th us
amphoteric molecules are located in narrow bands where the pI coincides with the pH. In this technique the point of applicati on is not
critical as molecules will always move to their pI region. The stable pH gradient between the electrodes is achieved using a mixture of
low molecular weight ampholytes which pI covers a preset range of pH.
Figure 3.14. Isoelectric Focusing. A pH gradient is established in a gel before loading the sample. After the sample is loaded a
voltage is applied. The protein will migrate to their isoelectric pH, which they have no net charge.
Two-Dimensional Gel Electrophoresis

Two-dimensional gel electrophoresis (2-DE) is based on separating a mixture of proteins according to two molecular properties, one in
each dimension. The most used is based on a first dimension separation by isoelectric focusing and second dimension according to
molecular weight by SDS-PAGE (Figure 3.15).
The general workflow in a 2-DE experiment would be:
• Sample Preparation
The method of sample preparation depends on the aim of the research and is crucial to the success of the experiment. Factors such as
the solubility, size, charge, and isoelectric point (pI) of the proteins of interest enter into sample preparation. Sample pr eparation is
also important in reducing the complexity of a protein mixture. The protein fraction to be loaded on a 2 -DE gel must be in a low ionic
strength denaturing buffer that maintains the native charges of proteins and keeps them soluble.
• First-Dimension Separation
This part is performed by IEF. Using this technique, proteins are separated on the basis of their pI, the pH at which a prote in carries
no net charge and will not migrate in an electrical field.
• Equilibration
A conditioning step is applied to proteins separated by IEF prior to the second -dimension run. This process reduces disulfide bonds
and alkylates the resultant sulfhydryl groups of the cysteine residues. Concurrently, proteins are coated with SDS for separa tion on
Proteins Page 32
and alkylates the resultant sulfhydryl groups of the cysteine residues. Concurrently, proteins are coated with SDS for separa tion on
the basis of molecular weight.
• Second-Dimension Separation
This part is performed by SDS-PAGE. The choice for the gel depends on the protein molecular weight range to be separated. The
ability to run many gels at the same time and under the same conditions is important for the purpose of gel -to-gel comparison.
• Staining
In order to visualize proteins in gels, they must be stained in some manner. The selection of staining method is determined b y several
factors, including desired sensitivity, linear range, ease of use, expense, and the type of imaging equipment available. At p resent there
is no ideal universal stain. Sometimes proteins are detected after transference to a membrane support by western blotting, wh ich is
described in more detail below.
• Image Analysis
The ability to collect data in digital form is one of the major factors that enable 2 -DE gels to be a practical means of collecting
proteome information. It allows unprejudiced comparison of gels and cataloging of immense amounts of data. Many types of imag ing
devices interface with software designed specifically to collect, interpret, and compare proteomics data. One of the biggest problems
in 2-DE is the analysis and comparison of complex mixtures of proteins. Currently there are databases capable of comparing two -
dimensional gel patterns. These systems allow automatic comparison of spots for the precise identification of those needed in the
quantitative analysis.
• Protein Identification
Once interesting proteins are selected by differential analysis or other criteria, the proteins can be excised from gels, dis tained and
digested to prepare their identification by mass spectrometry. This technique is known as peptide mass fingerprinting. The ab ility to
precisely determine molecular weight by matrix-assisted laser desorption/ionization- time of flight mass spectrometry (MALDI-TOF
MS) and to search databases for peptide mass matches has made high -throughput protein identification possible. Proteins not
identified by MALDI- TOF can be identified by sequence tagging or de novo sequencing using the Q -TOF electrospray LC-MS-MS.
Fig. 3.15 Two-Dimentional Gel Electrophoresis. Proteins of Chlamydomonas reinhardtii resolved by 2-DE from preparative gels
stained with MALDI-MS compatible silver reagent for peptide mass fingerprinting analysis. First dimension: isoelectric focusing in a
3-11 pH gradient. Second dimension: SDS-PAGE in a 12% acrylamide (2.6% crosslinking) gel (1.0 mm thick). Numbered spots marked
with circle correspond to proteins compared to be subsequently identified by MALDI -TOF MS. The MALDI-TOF MS analysis of
protein sequences is discussed in more detail in section 3.3 below.
back to the top
Antibody Structure and Production

(work derived fromCharles Molnar and Jane Gair)
An antibody, also known as an immunoglobulin (Ig), is a protein that is produced by plasma cells after stimulation by an antigen.
Proteins Page 33
An antibody, also known as an immunoglobulin (Ig), is a protein that is produced by plasma cells after stimulation by an antigen.
Antibodies are the functional basis of humoral immunity. Antibodies occur in the blood, in gastric and mucus secretions, and in breast
milk. Antibodies in these bodily fluids can bind pathogens and mark them for destruction by phagocytes before they can infect cells.
The molecule that is bound by an antibody is termed the antigen. Antibodies are highly specific for a single antigen or a group of
antigens that share highly conserved structural features. Proteins can act as antigens that are recognized by antibodies. Thu s, within
the field of biochemistry and molecular biology, antibodies are used as important tools that help us to determine the functio n and
expression pattern of proteins. They can also be used therapeutically in the treatment of diseases such as cancer.
Antibody Structure
The most common type of antibody used in biochemical methodologies is known as immunoglobulin G (IgG) and will be the focus o f this
section. An IgG antibody molecule is comprised of four polypeptides: two identical heavy chains (large peptide units) that ar e partially
bound to each other in a “Y” formation, which are flanked by two identical light chains (small peptide units), as illustrated in Figure
3.16. Bonds between the cysteine amino acids in the antibody molecule attach the polypeptides to each other. The areas where the
antigen is recognized on the antibody are variable domains and the antibody base is composed of constant domains.
Figure 3.16 Immunoglobulin G (IgG) Structure(a) As a germ-line B cell matures, an enzyme called DNA recombinase randomly excises
V and J segments from the light chain gene. Splicing at the mRNA level results in further gene rearrangement. As a result, (b ) each
mature B cell produces a single antibody that has a unique variable region capable of binding a different antigen.
Image from: Charles Molnar and Jane Gair
In germ-line B cells, the variable region of the light chain gene has 40 variable (V) and five joining (J) segments. An enzyme called
DNA recombinase randomly excises most of these segments out of the gene during B cell maturation, and splices one V segment t o one
J segment. During RNA processing, all but one V and J segment are spliced out. Recombination and splicing may result in over 106
possible VJ combinations! As a result, each differentiated B cell in the human body typically has a unique variable chain tha t will
recognize a unique antigen. The constant domain, which does not bind antibody, is the same for all antibodies.
Production of Polyclonal Antibodies
Antibodies used for research and diagnostic purposes are often obtained by injecting a lab animal such as a rabbit or a goat with a
specific antigen. Within a few weeks, the animal’s immune system will produce high levels of antibodies specific for the antigen. These
antibodies can be harvested in an antiserum, which is whole serum collected from an animal following exposure to an antigen. Because
most antigens are complex structures with multiple epitopes, they result in the production of multiple antibodies in the lab animal. This
so-called polyclonal antibody response is also typical of the response to infection by the human immune system. Antiserum drawn from
an animal will thus contain antibodies from multiple clones of B cells, with each B cell responding to a specific epitope on the antigen
Proteins Page 34
an animal will thus contain antibodies from multiple clones of B cells, with each B cell responding to a specific epitope on the antigen
(Figure 3.17).
Figure 3.17. Polyclonal Antibody Production. This diagram illustrates the process for harvesting polyclonal antibodies produced in
response to an antigen.
Image from Polyclonal and Monoclonal Antibody Production
Lab animals are usually injected at least twice with antigen when being used to produce antiserum. The second injection will activate
memory cells that make class IgG antibodies against the antigen. The memory cells also undergo affinity maturation, resulting in a pool
of antibodies with higher average affinity. Affinity maturation occurs because of mutations in the immunoglobulin gene variab le
regions, resulting in B cells with slightly altered antigen-binding sites. On re-exposure to the antigen, those B cells capable of
producing antibody with higher affinity antigen-binding sites will be stimulated to proliferate and produce more antibody than their
lower-affinity peers. An adjuvant, which is a chemical that provokes a generalized activation of the immune system that stimulates
greater antibody production, is often mixed with the antigen prior to injection.
Antiserum obtained from animals will not only contain antibodies against the antigen artificially introduced in the laborator y, but it will
also contain antibodies to any other antigens to which the animal has been exposed during its lifetime. For this reason, anti sera must
first be “purified” to remove other antibodies before using the antibodies for research or diagnostic assays.
Production of Monoclonal Antibodies
Some types of assays require better antibody specificity and affinity than can be obtained using a polyclonal antiserum. To a ttain this
high specificity, all of the antibodies must bind with high affinity to a single epitope. This high specificity can be provid ed by
monoclonal antibodies (mAbs). Table 3.4 compares some of the important characteristics of monoclonal and polyclonal antibodies.
Table 3.4 Comparison of Monoclonal and Polyclonal Antibodies
Unlike polyclonal antibodies, which are produced in live animals, monoclonal antibodies are produced in vitro using tissue-culture
techniques. mAbs are produced by immunizing an animal, often a mouse, multiple times with a specific antigen. B cells from th e spleen
of the immunized animal are then removed. Since normal B cells are unable to proliferate forever, they are fused with immorta l,
cancerous B cells called myeloma cells, to yield hybridoma cells. All of the cells are then placed in a selective medium that allows only
the hybridomas to grow; unfused myeloma cells cannot grow, and any unfused B cells die off. The hybridomas, which are capable of
growing continuously in culture while producing antibodies, are then screened for the desired mAb. Those producing the desire d mAb
are grown in tissue culture; the culture medium is harvested periodically and mAbs are purified from the medium. This is a ve ry
expensive and time-consuming process. It may take weeks of culturing and many liters of media to provide enough mAbs for an
experiment or to treat a single patient. mAbs are expensive (Figure 3.18).
Proteins Page 35
Figure 3.18. Monoclonal Antibodies (mAbs) are produced by introducing an antigen to a mouse and then fusing polyclonal B cells from
the mouse’s spleen to myeloma cells. The resulting hybridoma cells are cultured and continue to produce antibodies to the ant igen.
Hybridomas producing the desired mAb are then grown in large numbers on a selective medium that is periodically harvested to obtain
the desired mAbs.
Image fromPolyclonal and Monoclonal Antibody Production
back to the top
Enzyme-Linked Immunosorbent Assay (ELISA) and Microarrays

(work derived fromthe Human Atlas Project)
Since the very first use of antibodies for the detection of antigens, many different technologies have been developed that ma ke use
of the antibodies’ capability to bind to other molecules. During the 1950s, the scientists Yalow and Berson developed a metho d where
radioactivity is used to determine the amount of an analyte in a solution. This so called ‘radioimmunoassay’ (RIA), for which Yarlow
received the Nobel prize in 1977, was a very sensitive method for the detection of hormones but using radioactivity for antig en
detection is not safe and suitable for a general use. Hence, an alternative procedure was developed by linking enzymes to ant ibodies
instead of a radioactive molecule, and by adhering molecules to surfaces. In one of the nowadays most common applications tod ay are
measuring the quantity of a biomolecule in a sample by “enzyme-linked immunosorbent assay” (ELISA). This term originally refers to
the use of an enzyme to report an interaction between an antibody and its binding partner (Gan & Patel, 2013). The foundation for
Perlmann and Engvall in Sweden (Engvall & Perlmann, 1971)as well as Schuurs and van Weemen from the Netherlands (Van Weemen &
Schuurs, 1971), who built assays with immobilized and enzyme-modified reagents in the early 1970s. Today, scientists also use colored
molecules (so called fluorophores) that re-emit light upon excitation to visualize antibody-antigen interactions. Many variants of
experimental procedures have been developed, and it is common to build assays using more than one antibody to detect a target of
interest (see Figure 3.xx C-D). To further enhance the possibilities offered by the immunoassay format, applications based on
microarrays have been developed and which allow to measure more than one molecule in a single reaction chamber (see below).
ELISA Assay Design
The use of antibodies allows designing experiments in many different ways for the intended analysis. To achieve the best poss ible
results from the experiment, different reagents, additives, and solutions have to be tested for their optimal combination and
concentration, incubation times and the number of wash cycles need to be evaluated and adjusted. This is to avoid unwanted
interactions, which disturb the analysis from detecting the target of interest. Moreover, the mode of how a target is identif ied and
detection can be performed in a number of ways, as described in Figure 3.19
Proteins Page 36
Figure 3.19. Different setups for ELISA and Other Immunossays. In ELISA assays, the antibodies may (A) detect an immobilized
antigen, (B) capture a labeled antigen, (C) capture an unlabeled antigen and use a second, labeled antibody to detect the cap tured
antigen, or (D) use a third antibody for detection, or even use two antibodies for detection (E). Direct labeling of the anti body or
antigen as in (A), (B), and (C) is the simplest and fastest method for detection. Using a secondary antibody as detection met hod, as
shown in (D) and (E), will further increase the sensitivity and selectivity of the analysis. The method used in (D) also allo ws greater
flexibility, whereas method (E) further increases the specificity, as three antibodies must bind the antigen in order to prod uce a
reporter molecule. Out of the presented assays, the most commonly used concepts are shown in (C) and (D).
Image from The Human Atlas Project
Multiplexing
A new era in immunoassays started with the development of a technology called microarrays. The term microarray most commonly
describes the ordered organization of small volume droplets that have dried on a small surface area. The reaction dimensions are
miniaturized so that many assays can be performed in multiple samples in parallel, several thousands of different features ma y be
presented to the surrounding solution. This means that scientists can measure a large number of molecules with one single exp eriment.
There is the possibility to use microscope glass slides and specialized robotics that deposit very small drops of liquid (1 n l =
0.000000001 liter) on the glass surface in an ordered fashion. This leaves behind spots of less than one millimeter in diamet er (0.15
mm). Another common technique for multiplexing is to use even smaller and color -coded particles (diameter of 0.005 mm). These
particles can be coated with antibodies to fish out the analyte from the solution.
Sensitivity
In many applications it is important to measure very small amounts (sometimes only traces) of a molecule in a given sample. I n order to
achieve the required sensitivity, the conditions of the experiment need to be adjusted to suit the antibodies, the detection system,
and the type of samples. In addition, there is progress being made on using better colors,signal amplification specialized la sers and
filters, as well as miniaturization(Ekins & Edwards, 1997).
Specific examples
There are many examples of how ELISA assays may be used in basic research and in clinical diagnostics. One specific example i s the
sensitive sandwich-type enzyme-linked immunoassay used to determine the amount of the protein prostate-specific antigen (PSA),
which is a biomarker used to detect prostate cancer(Kuriyama et al., 1980).
Microarray assays on the other hand, have previously received a lot of attention for their use in parallel analysis of DNA an d RNA
molecules. To translate their advantages to assays for the analysis of proteins with antibodies, new protocols and routines h ad to be
developed and established. Nowadays, there are multiplexed techniques for measuring the amount of proteins in different sampl e
types (e.g. cells, blood serum, urine), to determine how proteins are modified in biological processes (e.g. phosphorylation) , or to
describe specific protein-protein interactions. Another example is the analysis of antibodies circulating in blood from patients.
Microarray-based applications have also been built for purified antibodies and to study the antibody binding characteristics – an
important aspect when using binding reagents as research reagents. Such protein microarrays can either consist of proteins, p rotein
fragments, or small peptides to test the specificity of the binding reagent. Protein microarrays can reveal the interactions to entire
proteins or larger protein fragments, while peptide microarrays show to which particular parts (epitopes) of the proteins the
antibodies bind. A typical epitope mapping result is shown in Figure 3.20 (Edfors et al., 2014). Synthesizing millions of overlapping
peptides with only one amino acid residue shift on such arrays enables the mapping of antibody binding regions at high resolu tion. This
gives very detailed information of the linear (continuous) epitopes recognized by an antibody. Just like with proteins, prote in
fragments or other antigens, the assembly of peptides on arrays may also be used for studies of antibody reactivity in plasma samples
from patients with infectious and autoimmune diseases.
Figure 3.20. Epitope Mapping of Polyclonal Antibodies. Polyclonal antibodies binding to a peptide array where the result displays
four distinct linear epitopes and the consecutive overlapping peptides which are bound. X -axis: peptides, Y-axis: mean fluorescence
Proteins Page 37
four distinct linear epitopes and the consecutive overlapping peptides which are bound. X -axis: peptides, Y-axis: mean fluorescence
intensity (MFI).(Edfors et al., 2014)
back to the top
Western Blot
Western Blot (WB) is a common method to detect and analyze proteins. It is built on a technique that involves transferring, a lso known
as blotting, proteins separated by electrophoresis from the gel to a membrane where they can be visualized specifically. The
procedure was first described by H. Towbin et al in 1979(Towbin, Staehelin, & Gordon, 1979) and two years later given its name by W.
Neal Burnette(Burnette, 1981). Towbin et al described electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose
sheets where the original gel pattern was accurately obtained. The setup consists of a standard set of seven steps, Figure 3. 21.
Figure 3.21. The Standard Steps in Western Blotting. The standard steps involve sample preparation (1), Gel electrophoresis (2),
Blotting to membrane (3), Antibody probing (4), Detection (5), Imaging (6) and Analysis (7).
Image fromthe Human Atlas Project.
Samples are prepared and loaded on to a gel and during the electrophoresis the negatively charged proteins move toward the po sitively
charged anode. In order to further analyze the proteins, they are transferred onto a membrane in a procedure called blotting. After
the transfer, the membrane is blocked in order to prevent unwanted membrane -protein interaction in the following steps. To visualize
the protein of interest the membrane is commonly first probed using a primary protein -specific antibody followed by a labeled
secondary antibody used for detection. An image is taken of the membrane and the result is analyzed.
By adding a separate marker solution to one of the wells in the gel, it is possible to estimate the size of the protein in ad dition to the
antibody interactions that are used to verify the specific protein. The separation on the gel is not only due to size but als o to some
extent depending on the molecular charge, hydrophobic regions, and degree of denaturation. The setup of the experiment can be
varied in many ways to best suit the specific inquiry. When analyzing the results, variations between lanes regarding loading and
transfer rates between blots, must be taken into consideration. In addition, the non -linear relation of the generated signal across the
concentration range of the samples is also an aspect of consideration when interpreting the results. The outcome of a WB expe riment
depends on three important factors; the ability of the antibody to bind a specific protein, the strength of the interaction, and the
concentration of the protein of interest itself. Moreover, the specificity of the binding to the target and a low cross react ivity are
important features as well. The result form the WB is not always easy to interpret as the size of the protein may vary from t he
theoretical weight due to posttranslational modifications, such as glycosylation, or interactions with other proteins. Howeve r, WB is a
very common method and almost all available commercial antibodies have been validatedusing this method.
Sample preparation
The first step of a WB is to prepare the sample, e.g. tissue, cells, or other solution, which is going to be analyzed. Usuall y the tissue
needs to be broken down by blending, homogenization, or sonication. Buffers are added to lyse the cells and solubilize the pr oteins and
often an inhibitor is added to prevent denaturation or degradation. Different types of filtration and centrifugation methods are
applied to further prepare the samples. It is important to determine the total protein concentration of the generated extract to be
able to load a specific amount on the gel to enable comparison between samples. Usually a biochemical assay is used in order to
determine the protein concentration. The extract is then diluted with loading buffer consisting of glycerol and a dye (e.g. b romophenol
blue). The glycerol is used to simplify the loading by raising the density of the extract and the dye is added to visualize t he sample.
Heat is applied on the samples in order to break the structures of the protein, which will help keeping the negative charge f rom
neutralization(Mahmood & Yang, 2012). Preferably positive and negative controls are included in the set up to confirm identity of the
protein as well as the activity of the antibody.
Gel electrophoresis
After sample preparation the extract is ready to be loaded to separate the proteins according to size by gel electrophoresis. An
electric field is applied over the gel that causes the charged molecules to move. In WB polyacrylamide gels are used for prot ein
separation and the method is therefore called polyacrylamide gel electrophoresis (PAGE) when using native condition. For dena turing
conditions, sodium dodecyl sulfate (SDS) is added to the system and the method is therefore called SDS -PAGE. The SDS binds to the
protein and form a negatively charged micelle around the protein regardless of inherent charge. The denaturing condition diss olves the
tridimensional structure of the proteins and the charge of the protein becomes relative to its size resulting in separation o f the
proteins only by size. When using native conditions the mobility is depending on both charge and hydrodynamic size allowing d etection
of changes in charge due to chemical degradation, conformational changes due to folding or unfolding, aggregation, or other b inding
events.
The gel typically consists of two sections with different densities: (i) a stacking gel, and (ii) a separating gel,(Figure 3. 22). The
differences between the two sections are in pH and gel concentration. With somewhat acidic pH and a lower concentration of
acrylamide the stacking gel separates the proteins poorly but allows them to form highly defined sharp bands before they ente r the
separating gel. With more basic conditions and higher gel concentration, the separating gel makes the proteins differentiate by size as
smaller proteins travel faster in the gel than bigger ones. Precast gels are convenient; however, it is possible to cast them by hand.
The gel is immersed in buffer and the protein samples and markers are loaded to the wells in the gel. A voltage is applied on the gel
and the proteins will start to travel down the gel due to their negative electrical charge. Selecting the proper voltage is i mportant
since too high voltage will overheat the gel and maybe deform the bands.
Proteins Page 38
Figure 3.22. A Typical Vertical Polyacrylamide Gel in Buffer
Blotting to membrane
After gel electrophoresis the proteins are transferred to a solid support membrane, which is the third step of Western Blot. In the
transfer process voltage is applied to transfer the proteins from the gel to the membrane. The setup includes sponges, filter papers,
the gel, and the membrane, which is placed between the gel and the positive electrode (Figure 3.23). This ensures the migrati on of the
negatively charge proteins from the gel to the membrane. There are three types of membranes: nitrocellulose, polyvinylidene
difluoride (PVDF), and nylon. Even though nylon membranes are superior in several aspects, the high background binding and
irreversible staining of some dyes makes this type of membrane less common than the other two alternatives. The major advanta ge of
nitrocellulose membranes is the low background regardless of detection method. Due to a relatively large average pore size,
nitrocellulose membranes should not be used for transfer of proteins with low molecular weight. Moreover, when dry, the membr ane
becomes brittle which makes them difficult to handle. The more stable PVDF membrane allows relabeling and is more convenient to
store. The hydrophobic nature of PVDF result in high protein binding capacity, however, as a consequence the background is al so
higher.
Figure 3.23. Transfer Procedure for Western Blotting. The proteins in the gel are transferred to a membrane and the sample is
visualized through blocking, adding antibodies, and washing according to a certain schedule.
There are two methods for the blotting called wet and semi-dry. The wet conditions are preferred when the transfer must be
efficient and give high quality regarding distinct and sharp bands. In addition, this is the better choice for transfer of la rger protein
complex. The gel, membrane, and filter papers are completely immersed in buffer during the transfer and there is no risk of d rying
Proteins Page 39
complex. The gel, membrane, and filter papers are completely immersed in buffer during the transfer and there is no risk of d rying
out the gel. Semi-dry blotting is more rapid and less volume of buffer is needed. However, this transfer method is usually less
efficient, especially for larger proteins, and there is a risk of overheating and drying the gel when using extended transfer times.
Antibody probing
The forth step of the WB is antibody probing. In order to prevent unspecific binding of the antibodies to the membrane, rathe r than
binding specific to the protein of interest, a substance is used to block out the residual sites on the membrane. Common subs tances
used are dried non-fat milk, 5% Bovine Serum Albumin (BSA) diluted in Tris Buffered Saline Tween (TBST), normal goat serum, casein,
or fish gelatin(Mahmood & Yang, 2012). Milk is easy to get hold of and inexpensive, however not suitable for all detection labels. Fish
gelatin gives lower background but can mask some proteins as well as being a relatively expensive blocking buffer. BSA is ine xpensive,
whereas serum can contain immunoglobulins giving rise to cross-reactivity. Careful selection of the blocking agent is key since none of
the blocking buffers are ideal for all different antigen-antibody interactions. The blocking procedure consists of incubating the
membrane in the appropriate blocking buffer for an hour or longer. When using long incubation times, the blocking should be
performed at +4°C to rule out the risk of staining artifacts or background. Blocking is a delicate balance between reducing t he
background without decreasing the signal from the protein of interest.
The blocked membrane is thereafter incubated with the primary antibody. The antibody is diluted to a suitable concentration i n TBST,
phosphate buffered saline (PBS), or wash buffer. It is preferred to incubate the antibody with BSA if the antibody is going t o be re-
used. After washing the membrane, the membrane is incubated with the secondary antibody that binds to the primary antibody. T he
secondary antibody is labeled with a reporter. When using a polyclonal antibody as secondary antibody, it may give rise to so me
background. In the case of background staining, the secondary antibody may be pre -blocked with non-immune serum from the host it
was generated in. Optimization of the concentration of the secondary antibody is recommended due to quite extended variations
between antibodies as well as detection system used.
Detection
In the fifth step of a WB, the protein-antibody-antibody complex is detected on the membrane. There are several kinds of labeling of
the secondary antibody, e.g. enzymes, fluorophores, biotinylation, gold-conjugation, and radioisotopes, as exemplified in Figure 3.24.
Amongst enzymes the most common is HRP used together with chemiluminescent, chemifluorescent, or chromogenic substances. HRP
has a high substrate specificity giving low background, is stable, and inexpensive. In chemiluminescense the HRP enzyme catal yzes the
oxidation of luminol from the luminol peroxide detection reagent. The multi -step reaction generates light emission. Certain chemicals
like phenols can enhance the emitted light. A direct method is the use of fluorescence; the fluorophores emit light after bei ng excited
and no detection agent is needed. It is well suitable for quantitative Western and since different fluorophores emit light of different
wavelengths it is possible to perform multiplexing and specific detection of more than one protein at the time. Using a chemi cal and/or
an enzyme to induce the generation of an active fluorophore from a fluorogenic substrate is called chemifluorescence. To furt her
enhance the signal intensity a two-step biotin streptavidin based system may be used. Gold conjugation is also a method where proteins
stain dark red due to accumulation of gold. It is also possible to use radioisotopes but they require special handling and ar e quite
expensive.
Figure 3.24. Different Reporter Systems.

Imaging
Imaging is the sixth step of WB and the capturing can be analogue using a film, or digitally preformed with a CCD camera or s canner
capturing the different kinds of emitted signals. The CCD imaging device enables quantitation with high detection sensitivity and a
broad linear range with no chemical waste or need for a dark room. It may be used to detect membranes, stained gels, or for
ultraviolet light applications.
Analysis
The last step of a WB is to analyze the results. In a typical qualitative application, the presence of a protein of interest is confirmed,
the amount is approximated by visual inspection, and the size is determined by comparison with a marker. Improvements and
developments, especially towards highly sensitive detection reagents and advanced imaging techniques, make WB a potential too l for
quantitative analysis. The quantitative applications entail a definition of the amount of protein in relative or absolute ter ms. Some
factors are to take under consideration like sensitivity, signal stability, linear dynamic range, normalization, and the sign al-to-noise
ratio. The minimum of protein that can be seen in a given assay gives the limits of detection (LOD), and the limit of signal intensity
that can be reliably used for precise quantification is the limit of quantification (LOQ). Factors that affect these terms ar e antibody
quality and concentrations as well as exposure times when considering the minimum amount of protein detected. A stable signal system
expands the time window for reaching high sensitivity, multiple exposures, and possibility to detect weak bands. The range th at allows
an even and precise quantitation where the signal intensity still is proportional to the amount of protein is called the line ar dynamic
range. It is important to avoid signal saturation due to excessive amounts of protein or high concentrations of antibodies. A low LOD
and quantitation of both weak and strong signals gives a broad linear dynamic range. The protein of interest should be normal ized to an
Proteins Page 40
and quantitation of both weak and strong signals gives a broad linear dynamic range. The protein of interest should be normal ized to an
internal reference that allows fluctuations in amount of protein loaded onto each well or different concentrations. This can be
achieved with housekeeping or spiked protein. The ratio between the signal and noise is important in order to properly quanti tate the
protein. This is of outmost importance when detecting weak bands where a higher background is expected. A typical Western Blo t is
seen in Figure 3.25.
Figure 3.25. Typical Western Blot result using HRP-conjugated antibodies and a CCD camera.
back to the top
Immunohistochemistry
Immunohistochemistry (IHC) is a powerful microscopy-based technique for visualizing cellular components, for instance proteins or
other macromolecules in tissue samples. The strength of IHC is the intuitive visual output that reveals the existence and loc alization
of the target-protein in the context of different cell types, biological states, and/or subcellular localization within complex tissues.
The IHC technique was invented during the 1940s(Coons, Creech, & Jones, 1941) and is routinely used as an important tool in health
care and pathology for e.g. diagnostic purposes or to stratify patients for optimized treatment regimes. IHC is also widely u sed in
research where molecules of interest are analyzed to study their roles in both healthy and diseased cells and tissues on the molecular,
cellular or tissue level. There are many different ways to perform visualization of targets in tissues using IHC or IHC -based methods,
and numerous protocols exist for different applications and assays. Even though IHC is generally a robust and established met hod, new
assays often need careful optimization depending on the tissue or on the properties of the target protein, binder -molecule and/or
reporter system. Many years of technical development and the hugely increased availability for specific binding -molecules have greatly
improved the usefulness and areas of applications for IHC. The progress in the field of IHC -based techniques and reagents has
enabled scientists and health care providers with more precise tools, assays and biomarkers. In addition, technical advances have
enabled e.g. highly sensitive simultaneous detection of multiple proteins in the same sample, and the detection of protein -protein
interactions.
The classical IHC assay is illustrated in Figure 3.26 and involves detection of epitopes expressed by a single protein -target within a
tissue sample using a “primary antibody” capable of binding those epitopes with high specificity. After the epitope -antibody binding
event, a “secondary antibody” capable of binding the primary antibody with high specificity is added. The secondary antibody is coupled
to a reporter molecule and after the antibody-antibody binding event, a chemical substrate is added which reacts with the reporter
molecule to produce a colored precipitate at the site of the whole epitope -antibody complex.
Figure 3.26. The Basic Principle of Immunohistochemistry. In the schematic illustration, a formalin-fixed paraffin embedded tissue
section is stained using a primary antibody directed towards a specific protein target. A solution containing the primary ant ibody is
Proteins Page 41
section is stained using a primary antibody directed towards a specific protein target. A solution containing the primary ant ibody is
added to the tissue section and the antibodies are allowed some time to find and bind to their target. After this step, unbou nd and
surplus antibodies are washed away and the secondary antibody is added. The secondary antibody, which carries a linker molecu le with
horseradish peroxidase (HRP) enzymes, is also allowed some time to bind to the primary antibody, followed by another washing step.
After this, 3,3′ Diaminobenzidine (DAB) is added. The HRP enzyme transforms the DAB substrate into a brownish precipitate that is
deposited in the tissue at the site of the reaction, thus producing a visual representation of where the primary antibody fir st bound
its target.
Tissue preparation
The tissue plays a central role in the experiment and it is important that it is processed so that epitopes and proper morpho logy is
preserved. The most common processing for IHC is to prepare formalin-fixed paraffin-embedded (FFPE) tissue blocks. The purpose of
formalin fixation is to produce chemical cross-linking of proteins within the tissue. This terminates all cellular processes and freezes
the cellular components at the place and in the conformation they were in at the time of fixation and also prevent degradatio n. After
adequate fixation, the tissue is further processed and ultimately embedded in paraffin blocks, which are then sectioned into thin
slices (usually 4-10µm) using a microtome. The sections are transferred to glass slides and allowed to adhere prior to further
processing.
Other methods for fixation besides formalin are sometimes used. These include other types of aldehydes or using different alc ohol
solutions. The best choice of fixative is very much dependent on the assay. A common alternative to FFPE is to prepare frozen tissue
samples. In this case, the tissue is embedded in a cryoprotective medium and frozen, and fixation is performed post -sectioning. Frozen
tissues are sectioned in cryostats and have the advantage of short processing times and of better preservation of sensitive e pitopes,
but can often be inferior to FFPE tissues in terms of preserving histological morphology.
Antigen (epitope) retrieval
A concern associated with cross-linking fixatives like formalin, or too long time spent in fixative medium is the masking of epitopes,
which can obstruct the primary antibody from binding to its target. Especially with FFPE samples, there is often a need to re vert some
of the chemical crosslinking and “retrieve” the epitopes before proceeding to the actual IHC. There are several antigen retri eval
protocols available and the main strategies include treating the tissue slide with heat, digestive enzymes, detergents, or co mbinations
thereof. The most common method for antigen retrieval in FFPE samples is to pressure -boil the tissue slides in an acidic citrate buffer
for around 15-20 minutes.
Antibody binding
The quality and specificity of the binding molecule is crucial for any IHC based technique, and the choice of binder can dire ctly affect
the outcome, reliability, and possibly also the interpretation of the assay. Antibodies are by far the most common type of bi nding-
molecule used for IHC, and although most antibodies are able to adequately detect the correct molecule of interest, they may also
vary greatly in their specificity for their intended target. Antibodies with high specificity are therefore more reliable whe n
interpreting “on-target” binding, since they produce little or no “off-target” binding or “background”. Antibodies that are less specific
can produce more off-target binding, and the resulting background will possibly interfere with the correct interpretation of the true
on-target signals. There are two main types of antibodies; polyclonal antibodies which is a heterogeneous mix of antibodies that bind
different epitopes on the target and monoclonal antibodies that all bind the same epitope. Polyclonal antibodies are often ve ry potent
due to their ability to detect and bind multiple epitopes on the same target. However, the epitopes they bind are often poorl y defined
and with multiple and varying epitope-specificities comes the increased likelihood of off-target binding events and background noise.
However, the potency of polyclonal antibodies can be advantageous since the concentration of binding events around the on -target
molecule usually outweighs potential background noise. A drawback is that polyclonal antibodies are usually limited resources since they
are derived from animal sera. Monoclonal antibodies, by contrast, have more continuity since they can be produced in hybridom a cell
lines. Monoclonal antibodies are also often well defined in terms of epitope binding, but can still generate results that are hard to
interpret if the specificity is low or if the target epitope is present in low abundance.
Careful optimization and titration of antibody concentration for each assay is needed, since the result is dependent not only on the
antibody’s specificity and affinity for the target, but also on the concentration and availability of on -target and potential off-target
epitopes present in the sample. Adding too much antibodies to the sample will increase the number of possible low -affinity off-target
binding events once the on-target epitope(s) are saturated with binders. By lowering the antibody concentration, off -target binding
events become rarer as they usually have lower affinity than on-target binding events. The risk when attempting to reduce background
while using a low-affinity antibody is that the on-target signals are concomitantly weakened to the point of providing a false negative
result.
Other types of binder molecules sometimes used in IHC-based techniques include affibodies, peptides, antibody fragments or other
small molecules.
Detection systems
The whole purpose of performing IHC is to obtain a visual representation of where the target can be found within the experime ntal
tissue, and preferably also gain information about the target’s expression pattern among heterogeneous cell populations and/o r
subcellular localizations. This is exemplified in Figure 3.27, which illustrates how different antibodies are used to visuali ze different
cellular or tissue compartments within a complex tissue. To visualize the target-antibody interaction, some kind of detection system
that produces an observable stain or signal is needed. The most common method for introducing a detection system to the exper iment
is to use a secondary antibody that carries a pre-bound reporter molecule, i.e. enzyme or fluorophore. Secondary antibodies are usually
targeted specifically towards antibody molecules from a different animal species. For example, if the primary antibody is rai sed in a
rabbit, then the secondary antibody must be raised in another animal and targeted specifically towards rabbit antibodies.
Proteins Page 42
Figure 3.27. Visualizing different protein targets in complex tissues. The right column shows a magnification of the corresponding
images in the left column. In the IHC image, consecutive sections of human esophagus stained using four different antibodies allows
for direct comparison of different protein expression patterns within the tissue and within subcellular compartments. The top images
are only counterstained for hematoxylin for comparison. The p63 antibody stains cell nuclei in a population of cells that res ide in the
basal part of the esophageal epithelium. The EGFR (Epidermal growth factor receptor) antibody appears to stain the same cell
population as p63, but stains cellular membranes instead of nuclei. The G6PD (Glucose -6-phosphate dehydrogenase) antibody stains the
cytoplasm of a wider repertoire of esophageal epithelial cells and also cells residing in the connective tissue. The Laminin (LAMB2)
antibody stains only cells and structures in the connective tissue underlying the esophagus.
For FFPE tissue samples the most common detection method is to use enzymatic reactions to generate a colored precipitate at t he site
of antibody binding. The secondary antibodies then carry an enzyme, e.g. horseradish peroxidase (HRP) or alkaline phosphatase (AP),
that are capable of converting chromogens like 3,3′ Diaminobenzidine (DAB) or 5-bromo-4-chloro-3-indolyl phosphate/ p-nitroblue
tetrazolium chloride (BCIP/NBT) into brown or bluish precipitates that are deposited in the tissue at the site of the reactio n.
Chromogenic stains are observable in light-microscopy and are usually very stable over long periods of time, which is beneficial if the
experiment needs to be archived or reviewed at a later time point.
For frozen tissue sections it is more common to use fluorophore-linked secondary antibodies that emit a specific color (usually green,
red, or blue) when excited by the correct wavelengths of light. Moreover, fluorophores are usually not stable for long period s of time.
However, the benefit of using fluorophores is that they provide an easy method for performing double -labeling experiments where
Proteins Page 43
However, the benefit of using fluorophores is that they provide an easy method for performing double -labeling experiments where
several antibodies towards multiple targets are assayed in the same sample. The secondary antibodies need to be targeted towa rds
different primary antibodies and also to be coupled to different fluorophores. The different secondary antibodies are then ob served
separately by exciting them sequentially with different wavelengths of light. These different excitation results are saved as separate
images (or color channels) and may later be overlaid to infer protein co-localizations etc.
Using reporter-carrying secondary antibodies for detection is in itself an amplification step since several secondary antibodies are
able to bind a single primary antibody, but sometimes further amplification steps are desired to increase the signal and sens itivity of
the experiment. In such cases, the secondary antibody may instead carry “linker molecules”, for instance biotin polymers, whi ch are
able to recruit a larger number of reporter molecules in subsequent steps. This strategy for amplifying signals is useful for both
enzymatic and fluorescent detection methods.
Counterstaining
Immunohistochemical staining using chromogens often benefits from having a counterstain applied that enhances the contrast an d
facilitates the observation of histological features. The most common type of counterstain used for FFPE samples is hematoxyl in that
stains cellular cytoplasm with a pale bluish color, and stain cell nuclei in a darker bluish nuance, as shown in Figure 3.26. Fluorescent
stainings are usually not counterstained with hematoxylin, since the detection method is not based on light microscopy. Inste ad, the
most common way to obtain counterstaining for fluorescence is to label cell nuclei by adding fluorescent dyes that bind nucle ic acids,
as shown in Figure 3.28. After the actual immunohistochemical reaction, the only remaining steps are to coverslip and seal th e sample
for protection and longterm storage. The most common way is to “glue” the coverslip to the sample using commercially availabl e
purpose-made resins.
Figure 3.28 Endothelial cells under the microscope.Nuclei are stained blue with DAPI, microtubles are marked green by an antibody
bound to FITC and actin filaments are labelled red with phalloidin bound to TRITC. Bovine pulmonary artery endothelial cells
Image from NIH ImageJ-Programmpaket
Specific examples
IHC is widely used in both research and clinical practice.The Human Protein Atlas (HPA) projectis a prime example of how high-
throughput IHC is used to achieve large-scale mapping of the human proteome in a multitude of tissues, cancers and cells. In the HPA
project, a streamlined in-house large scale antibody production chain facilitates the generation of specific antibodies, which after
passing basic characterization and validation regimes, are used to systematically stain tissue microarrays containing hundred s of tissue
cores within a single experiment. The system for IHC employed by HPA relies heavily on standardization of protocols and
automatisation using machines, but the evaluation of the optimal titration for each antibody is performed manually before the antibody
is approved for staining on the full set of tissues. Each stained tissue core is annotated with respect to immunohistochemica l staining
in tissues and cell types, and thereafter published as a high resolution image on the web portal to be freely viewed by anyon e.
In clinical practice, IHC is mainly used within pathology to aid physicians to evaluate tissue specimens with respect to heal thy and or
diseased states, to set diagnoses, and to define the molecular subtype of different types of cancer. A specific example where IHC is
used diagnostically is when pathologists are presented with a metastatic tumor sample and the tissue origin of the primary tu mor is
unknown. In these cases, pathologists use a panel of different antibodies that target tissue specific proteins, such as prost ate-
specific antigen for prostate cancer, or estrogen receptor for gynecological cancers, or cytokeratin 20 for gastrointestinal
cancers(Gremel et al., 2014). Once a broad classification is made, additional tissue specific antibodies are used to further pinpoint the
origin of the primary tumor. This information is useful for choosing the best or most appropriate strategy for drug therapy a nd/or to
locate the primary tumor for radiation therapy and/or surgery.
back to the top
Proteins Page 44
Solid-Phase Protein Synthesis
Peptides are chemically synthesized by the condensation reaction of the carboxyl group of one amino acid to the amino group o f
another. Protecting group strategies are usually necessary to prevent undesirable side reactions with the various amino acid side
chains. Chemical peptide synthesis most commonly starts at the carboxyl end of the peptide (C -terminus), and proceeds toward the
amino-terminus (N-terminus). Protein biosynthesis (long peptides) in living organisms occurs in the opposite direction. Chemical
synthesis facilitates the production of peptides which are difficult to express in bacteria, the incorporation of unnatural a mino acids,
peptide/protein backbone modification, and the synthesis of D-proteins, which consist of D-amino acids.
The established method for the production of synthetic peptides in the lab is known as solid -phase peptide synthesis (SPPS).
Pioneered by Robert Bruce Merrifield, SPPS allows the rapid assembly of a peptide chain through successive reactions of amino acid
derivatives on an insoluble porous support.
The solid support consists of small, polymeric resin beads functionalized with reactive groups (such as amine or hydroxyl gro ups) that
link to the nascent peptide chain. Since the peptide remains covalently attached to the support throughout the synthesis, exc ess
reagents and side products can be removed by washing and filtration. This approach circumvents the comparatively time -consuming
isolation of the product peptide from solution after each reaction step, which would be required when using conventional solu tion-
phase synthesis.
Each amino acid to be coupled to the peptide chain N-terminus must be protected on its N-terminus and side chain using appropriate
protecting groups such as Boc (acid-labile) or Fmoc (base-labile), depending on the side chain and the protection strategy used (FIg.
3.29).
The general SPPS procedure is one of repeated cycles of alternate N-terminal deprotection and coupling reactions. The resin can be
washed between each steps. First an amino acid is coupled to the resin. Subsequently, the amine is deprotected, and then coup led with
the free acid of the second amino acid. This cycle repeats until the desired sequence has been synthesized. SPPS cycles may a lso
include capping steps which block the ends of unreacted amino acids from reacting. At the end of the synthesis, the crude pep tide is
cleaved from the solid support while simultaneously removing all protecting groups using a reagent strong acids like trifluor oacetic acid
or a nucleophile. The crude peptide can be precipitated from a non-polar solvent like diethyl ether in order to remove organic soluble
by products. The crude peptide can be purified using reversed-phase HPLC. The purification process, especially of longer peptides can
be challenging, because small amounts of several byproducts, which are very similar to the product, have to be removed. For t his
reason so-called continuous chromatography processes such as MCSGP are increasingly being used in commercialsettings to maximize
the yield without sacrificing on purity levels.
SPPS is limited by reaction yields, and typically peptides and proteins in the range of 70 amino acids are pushing the limits of synthetic
accessibility. Synthetic difficulty also is sequence dependent; typically aggregation -prone sequences such as amyloids are difficult to
make. Longer lengths can be accessed by using ligation approaches such as native chemical ligation, where two shorter fully
deprotected synthetic peptides can be joined together in solution.
Figure 3.29 Solid-phase synthesis of a dipeptide using an (amine-functionalized) amide resin. The N-terminal protecting group
(PG) can be Fmoc or Boc, depending on the protecting group scheme used. The amino acid side chains (R 1, R2 etc.) are orthogonally
protected.
Image by Bédard, F. and Biron, E. (2018) Frontiers in Microbiology 9:1048
Protein Sequencing using Edman Degradation

Edman degradation, developed by Pehr Edman, is a method of sequencing amino acids in a peptide (Fig. 3.30). In this method, the
amino-terminal residue is labeled and cleaved from the peptide without disrupting the peptide bonds between other amino acid
residues.
Proteins Page 45
Figure 3.30 Edman Degradation Scheme. In this method, Phenyl isothiocyanate is reacted with an uncharged N-terminal amino group,
under mildly alkaline conditions, to form a cyclical phenylthiocarbamoyl derivative. Then, under acidic conditions, this derivative of the
terminal amino acid is cleaved as a thiazolinone derivative. The thiazolinone amino acid is then selectively extracted into a n organic
solvent and treated with acid to form the more stable phenylthiohydantoin (PTH) - amino acid derivative that can be identified by using
chromatography or electrophoresis. This procedure can then be repeated again to identify the next amino acid.
Image fromChoij
A major drawback to Edman degradation is that the peptides being sequenced in this manner cannot have more than 50 to 60 resi dues
(and in practice, under 30). The peptide length is limited due to the cyclical derivatization not always going to completion. The
derivatization problem can be resolved by cleaving large peptides into smaller peptides before proceeding with the reaction. It is able
to accurately sequence up to 30 amino acids with modern machines capable of over 99% efficiency per amino acid. An advantage of the
Edman degradation is that it only uses 10 – 100 pico-moles of peptide for the sequencing process. The Edman degradation reaction was
automated in 1967 by Edman and Beggs to speed up the process and 100 automated devices were in use worldwide by 1973.
Because the Edman degradation proceeds from the N-terminus of the protein, it will not work if the N-terminus has been chemically
modified (e.g. by acetylation or formation of pyroglutamic acid). Sequencing will stop if a non -α-amino acid is encountered (e.g.
isoaspartic acid), since the favored five-membered ring intermediate is unable to be formed. Edman degradation is generally not useful
to determine the positions of disulfide bridges. It also requires peptide amounts of 1 picomole or above for discernible resu lts.
Following 2D SDS PAGE the proteins can be transferred to a polyvinylidene difluoride (PVDF) blotting membrane for further ana lysis.
Edman degradations can be performed directly from a PVDF membrane. N-terminal residue sequencing resulting in five to ten amino
acid may be sufficient to identify a Protein of Interest (POI).
Sequence analysis by Matrix-Assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF) Mass Spectrometry.
Mass spectrometry is a technique to analyze with high accuracy the composition of different chemical elements and atomic isot opes
splitting their atomic nuclei according to their mass- charge ratio (m/z). It can be used to identify different chemical elements that
form a compound or to determine the isotopic content of different elements in the same compound.
Protein mass spectrometry refers to the application of mass spectrometry to the study of proteins. Mass spectrometry is an
important method for the accurate mass determination and characterization of proteins, and a variety of methods and
instrumentations have been developed for its many uses. Its applications include the identification of proteins and their pos t-
translational modifications, the elucidation of protein complexes, their subunits and functional interactions, as well as the global
measurement of proteins in proteomics. It can also be used to localize proteins to the various organelles, and determine the
interactions between different proteins as well as with membrane lipids.
Two techniques are often used with liquid and solid biological samples: electro spray ionization and laser matrix - assisted laser
desorption/ionization (MALDI). In the MALDI ionization analytes co- crystallized with a suitable matrix are converted into ions by the
action of a laser. This source of ionization is usually associated with a time of flight analyzer (TOF) in which the ions are separated
according to their mass-charge after being accelerated in an electric field. At last, a mass spectrometer detector records the charge
induced or current produced when an ion passes by or hits a surface. A mass spectrum is recorded for each protein (Figure 3.3 1).
Figure 3.31. Protein identification by MALDI-TOF MS. Workflow of protein identification developing MALDI-TOF MS assay (A),
followed by MS/MS fragmentation of peptides (B) and analysis of spectral data with the MASCOT database search algorithm (C).
In general, proteins are analyzed either in a “top-down” approach in which proteins are analyzed intact, or a “bottom-up” approach in
which protein are first digested into fragments. An intermediate “middle-down” approach in which larger peptide fragments are
analyzed may also sometimes be used. The top-down approach however is mostly limited to low-throughput single-protein studies due
to issues involved in handling whole proteins, their heterogeneity and the complexity of their analyses.
Proteins Page 46
to issues involved in handling whole proteins, their heterogeneity and the complexity of their analyses.
In the second approach, referred to as the “bottom-up” MS, proteins are enzymatically digested into smaller peptides using a protease
such as trypsin. Trypsin is a serine protease from the PA clan superfamily, found in the digestive system of many vertebrates, where
it hydrolyzes proteins. Trypsin cleaves peptide chains mainly at the carboxyl side of the amino acids lysine or arginine, exc ept when
either is followed by proline. It is used for numerous biotechnological processes. The process is commonly referred to as try psin
proteolysis or trypsinisation, and proteins that have been digested/treated with trypsin are said to have been trypsinized.
Subsequently, these peptides are introduced into the mass spectrometer and identified by peptide mass fingerprinting or tande m mass
spectrometry. Hence, this approach uses identification at the peptide level to infer the existence of proteins pieced back to gether
with de novo repeat detection. The smaller and more uniform fragments are easier to analyze than intact proteins and can be also
determined with high accuracy, this “bottom-up” approach is therefore the preferred method of studies in proteomics. A further
approach that is beginning to be useful is the intermediate “middle-down” approach in which proteolytic peptides larger than the
typical tryptic peptides are analyzed.
Proteins of interest are usually part of a complex mixture of multiple proteins and molecules, which co -exist in the biological medium.
This presents two significant problems. First, the two ionization techniques used for large molecules only work well when the mixture
contains roughly equal amounts of constituents, while in biological samples, different proteins tend to be present in widely differing
amounts. If such a mixture is ionized using electrospray or MALDI, the more abundant species have a tendency to “drown” or su ppress
signals from less abundant ones. Second, mass spectrum from a complex mixture is very difficult to interpret due to the overw helming
number of mixture components. This is exacerbated by the fact that enzymatic digestion of a protein gives rise to a large num ber of
peptide products.
In light of these problems, the methods of one- and two-dimensional gel electrophoresis and high performance liquid chromatography
are widely used for separation of proteins. The first method fractionates whole proteins via two -dimensional gel electrophoresis
(Figure 3.32). The first-dimension of 2D gel is isoelectric focusing (IEF). In this dimension, the protein is separated by its isoelectric
point (pI) and the second-dimension is SDS-polyacrylamide gel electrophoresis (SDS-PAGE). This dimension separates the protein
according to its molecular weight. Once this step is completed in-gel digestion occurs.
In some situations, it may be necessary to combine both of these techniques. Gel spots identified on a 2D Gel are usually att ributable
to one protein. If the identity of the protein is desired, usually the method of in -gel digestion is applied, where the protein spot of
interest is excised, and digested proteolytically. The peptidemasses resulting from the digestion can be determined by mass
spectrometry using peptide mass fingerprinting. If this information does not allow unequivocal identification of the protein, its
peptides can be subject to tandem mass spectrometry for de novo sequencing. Small changes in mass and charge can be detected with
2D-PAGE. The disadvantages with this technique are its small dynamic range compared to other methods, some proteins are still
difficult to separate due to their acidity, basicity, hydrophobicity, and size (too large or too small).
The second method, high performance liquid chromatography is used to fractionate peptides after enzymatic digestion.
Characterization of protein mixtures using HPLC/MS is also called shotgun proteomics and MuDPIT (Multi -Dimensional Protein
Identification Technology). A peptide mixture that results from digestion of a protein mixture is fractionated by one or two steps of
liquid chromatography. The eluent from the chromatography stage can be either directly introduced to the mass spectrometer
through electrospray ionization, or laid down on a series of small spots for later mass analysis using MALDI.
Proteins Page 47
Figure 3.32 Schematic of Protein Fingerprinting by Mass Spectrometry. Protein mixtures are prepared from cell culture or tussie
samples and separated by gel electrophoresis. Single proteins are isolated and digested using trypsin to produce a peptide mi xture.
Peptides are separated by liquid chromatography and analyzed by mass spectrometry
Image by Philippe Hupé
back to the top

X-Ray Crystallography
Protein X-Ray Crystallography is a technique used to obtain the three-dimensional structure of a particular protein by X-ray
diffraction of its crystallized form. This three dimensional structure is crucial to determining a protein’s functionality. M aking
crystals creates a lattice in which this technique aligns millions of proteins molecules together to make the data collection more
sensitive. It’s like getting a stack of papers, measuring the width with a ruler, and dividing that length with the number of pages to
determine the width of one piece of paper. By this averaging technique, the noise level gets reduced and the signal to noise ratio
increases. The specificity of the protein’s active sites and binding sites is completely dependent on the protein’s precise c onformation.
In addition to protein structure, other biological molecules can be investigated using X -ray crystallography. It was the X-ray
crystallography by Rosalind E.Franklin, that made it possible for J.D. Watson and F.H.C. Crick to figure out the double -helix structure
of DNA.
X-ray crystallography can reveal the detailed three-dimensional structures of thousands of proteins. The three components in an X -
ray crystallographic analysis are a protein crystal, a source of X-rays, and a detector.
X-ray crystallography is used to investigate molecular structures through the growth of solid crystals of the molecules they st udy.
Crystallographers aim high-powered X-rays at a tiny crystal containing trillions of identical molecules. The crystal scatters the X -rays
onto an electronic detector. The electronic detector is thesame type used to capture images in a digital camera. After each b last of
X-rays, lasting from a few seconds to several hours, the researchers precisely rotate the crystal by entering its desired orien tation
into the computer that controls the X-ray apparatus. This enables the scientists to capture in three dimensions how the crystal
scatters, or diffracts, X-rays. The intensity of each diffracted ray is fed into a computer, which uses a mathematical equation to
calculate the position of every atom in the crystallized molecule. The result is a three -dimensional digital image of the molecule (Figure
3.33).
Crystallographers measure the distances between atoms in angstroms. The perfect “rulers” to measure angstrom distances are X -rays.
The X-rays used by crystallographers are approximately 0.5 to 1.5 angstroms long, which are just the right size to measure the
distance between atoms in a molecule. If the radiation had a wavelength much bigger or much smaller than the bond length of a
covalent bond, the light would not diffract and no new knowledge of the structure would be obtained. That is why X -rays are used.
Proteins Page 48
Figure 3.33 Workflow for Solving the Structure of a Molecule by X-ray Crystallography
Image fromThomas Splettstoesser
The process begins by crystallizing a protein of interest. Crystallization of protein causes all the protein atoms to be orie ntated in a
fixed way with respect to one another while still maintaining their biologically active conformations – a requirement for X-ray
diffraction. A protein must be precipitated out or extracted from a solution. The rule of thumb here is to get as pure a prot ein as
possible to grow lots of crystals (this allows for the crystals to have charged properties, and surface charged distribution for better
scattering results). 4 critical steps are taken to achieve protein crystallization, they are:
1. Purify the protein. Determine the purity of the protein and if not pure (usually >99%), then must undergo further purification.
2. Must precipitate protein. Usually done so by dissolving the protein in an appropriate solvent(water-buffer soln. w/ organic salt
such as 2-methyl-2,4-pentanediol). If protein is insoluble in water-buffer or water-organic buffer then a detergent such as
sodium lauryl sulfate must be added.
3. The solution has to be brought to supersaturation(condensing the protein from the rest of the solvent forming condensation
nuclei). This is done by adding a salt to the concentrated solution of the protein, reducing its solubility and allowing the protein to
form a highly organized crystal (this process is referred to as salting out). Other methods include batch crystallization, liquid-
liquid crystallization, vapor diffusion, and dialysis.
4. Let the actual crystals grow. Since nuclei crystals are formed this will lead to obtaining actual crystal growth.
The crystals are then bombarded with X-rays, the diffraction patterns recorded, and the structure is reconfigured from the
diffraction pattern using Fourier Transformation. Through the Fourier Transform, the electron density distribution is illustr ated as a
series of parallel shapes and lines stacked on top of each other (contour lines), like a terrain map. The mapping gives a thr ee-
dimensional representation of the electron densities observed through the x-ray crystallography. When interpreting the electron
density map, resolution needs to be taken into account. A resolution of 5Å – 10Å can reveal the structure of polypeptide chains, 3Å –
4Å of groups of atoms, and 1Å – 1.5Å of individual atoms. The resolution is limited by the structure of the crystal and for proteins is
about 2Å.
Many advances in drug discovery and medicine are due in large part by X -Ray Crystallography by identifying drug targets in many
diseases that thrive today. In the late 80’s for example, scientists made a breakthrough in using X -Ray Crystallography to produce the
structure of HIV Protease, an enzyme that was vital to the retrovirus’ life cycle. The enzyme cuts viral proteins strands tha t are main
components of immature viral cells into separate, mature proteins that can continue on to form more mature and infectious vir al
particles. By looking closely at it structure, specifically its symmetry, researchers began making compounds that interacted with the
active site of the enzyme, which is in the middle of its symmetric halves, to shut the enzyme down and prevent it from functi oning
properly. Amazingly, by the mid 90s, three HIV Protease inhibitor drugs were on the market, drastically reducing the death ra te of
the AIDS Virus (Figure 3.34)
Proteins Page 49
Figure 3.34. Crystal structure of the D-enantiomer of backbone engineered HIV-1 PR prepared by totalchemical synthesis
complexed to D-MVT101 inhibitor. (a) Cocrystals were obtained from50% ammonium sulfate, pH 5.4, in the space group P212121a =
67.5, b = 92.8, c = 29.4 Å.There were two monomers of protein and one inhibitor in the crystallographic asymmetricunit. (b) S tereo
view of Cα tracing of D HIV-1 PR. Bound D MVT-101 inhibitor is shown asgreen sticks. Coordinates were deposited to HIV Structural
Database;48 accession code:NCI2009
Image from:Miller, M. (2010) Biopolymers 94(4):521-529.
back to the top
Nuclear Magnetic Resonance (NMR) Imaging

Nuclear magnetic resonance spectroscopy of proteins (usually abbreviated protein NMR) is a field of structural biology in which
NMR spectroscopy is used to obtain information about the structure and dynamics of proteins, and also nucleic acids, and thei r
complexes. The field was pioneered by Richard R. Ernst and Kurt Wüthrich at the ETH, and by Ad Bax, Marius Clore, and Angela
Gronenborn at the NIH, among others. Structure determination by NMR spectroscopy usually consists of several phases, each usi ng a
separate set of highly specialized techniques. The sample is prepared, measurements are made, interpretive approaches are app lied,
and a structure is calculated and validated.
NMR involves the quantum mechanical properties of the central core (“nucleus”) of the atom. These properties depend on the lo cal
molecular environment, and their measurement provides a map of how the atoms are linked chemically, how close they are in spa ce, and
how rapidly they move with respect to each other. These properties are fundamentally the same as those used in the more famil iar
magnetic resonance imaging (MRI), but the molecular applications use a somewhat different approach, appropriate to the change of
scale from millimeters (of interest to radiologists) to nanometers (bonded atoms are typically a fraction of a nanometer apar t). This
change of scale requires much higher sensitivity of detection and stability for long term measurement. In contrast to MRI, st ructural
biology studies do not directly generate an image, but rely on complex computer calculations to generate three -dimensional molecular
models.
Currently most samples are examined in a solution in water, but methods are being developed to also work with solid samples. Data
collection relies on placing the sample inside a powerful magnet, sending radio frequency signals through the sample, and mea suring the
absorption of those signals. Depending on the environment of atoms within the protein, the nuclei of individual atoms will ab sorb
different frequencies of radio signals. Furthermore, the absorption signals of different nuclei may be perturbed by adjacent nuclei.
This information can be used to determine the distance between nuclei. These distances in turn can be used to determine the o verall
structure of the protein.
A typical study might involve how two proteins interact with each other, possibly with a view to developing small molecules t hat can be
used to probe the normal biology of the interaction (“chemical biology”) or to provide possible leads for pharmaceutical use (drug
development). Frequently, the interacting pair of proteins may have been identified by studies of human genetics, indicating the
interaction can be disrupted by unfavorable mutations, or they may play a key role in the normal biology of a “model” organis m like the
fruit fly, yeast, the worm C. elegans, or mice.
Protein nuclear magnetic resonance is performed on aqueous samples of highly purified protein. Usually, the sample consists o f
between 300 and 600 microlitres with a protein concentration in the range 0.1 – 3 millimolar. The source of the protein can be either
natural or produced in a production system using recombinant DNA techniques through genetic engineering. Recombinantly expres sed
proteins are usually easier to produce in sufficient quantity, and this method makes isotopic labeling possible.
The purified protein is usually dissolved in a buffer solution and adjusted to the desired solvent conditions. The NMR sample is
prepared in a thin-walled glass tube. (Figure 3.35)
Proteins Page 50
Figure 3.35 The NMR Sample is Prepared in a Thin-Walled Glass Tube
Image fromKjaergaard
With unlabelled protein the usual procedure is to record a set of two dimensional homonuclear nuclear magnetic resonance expe riments
through correlation spectroscopy (COSY), of which several types include conventional correlation spectroscopy, total correlation
spectroscopy (TOCSY) and nuclear Overhauser effect spectroscopy (NOESY). A two -dimensional nuclear magnetic resonance
experiment produces a two-dimensional spectrum. The units of both axes are chemical shifts. The COSY and TOCSY transfer
magnetization through the chemical bonds between adjacent protons (Figure 3.36). The conventional correlation spectroscopy
experiment is only able to transfer magnetization between protons on adjacent atoms, whereas in the total correlation spectro scopy
experiment the protons are able to relay the magnetization, so it is transferred among all the protons that are connected by adjacent
atoms. Thus in a conventional correlation spectroscopy, an alpha proton transfers magnetization to the beta protons, the beta protons
transfers to the alpha and gamma protons, if any are present, then the gamma proton transfers to the beta and the delta proto ns, and
the process continues. In total correlation spectroscopy, the alpha and all the other protons are able to transfer magnetizat ion to the
beta, gamma, delta, epsilon if they are connected by a continuous chain of protons. The continuous chain of protons are the s idechain
of the individual amino acids.
Thus these two experiments are used to build so called spin systems, that is build a list of resonances of the chemical shift of the
peptide proton, the alpha protons and all the protons from each residue’s sidechain. Which chemical shifts corresponds to whi ch nuclei
in the spin system is determined by the conventional correlation spectroscopy connectivities and the fact that different type s of
protons have characteristic chemical shifts. To connect the different spinsystems in a sequential order, the nuclear Overhaus er
effect spectroscopy experiment has to be used. Because this experiment transfers magnetization through space, it will show
crosspeaks for all protons that are close in space regardless of whether they are in the same spin system or not. The neighbo uring
residues are inherently close in space, so the assignments can be made by the peaks in the NOESY with other spin systems.
Proteins Page 51
residues are inherently close in space, so the assignments can be made by the peaks in the NOESY with other spin systems.
Figure 3.36: Comparison of a COSY and TOCSY 2D spectra for an amino acid like glutamate or methionine. The TOCSY shows
off diagonal crosspeaks between all protons in the spectrum, but the COSY only has crosspeaks between neighbours.
Image from Kjaergaard
One important problem using homonuclear nuclear magnetic resonance is overlap between peaks. This occurs when different proto ns
have the same or very similar chemical shifts. This problem becomes greater as the protein becomes larger, so homonuclear nuc lear
magnetic resonance is usually restricted to small proteins or peptides.
If recombinant proteins can be produced, the resulting protein can be labelled with Nitrogen -15 or with Carbon-13 to allow for more
detailed experimentation, such as heteronuclear single quantum coherence spectroscopy (HSQC). The most commonly performed 15N
experiment is the 1H-15N HSQC. The experiment is highly sensitive and therefore can be performed relatively quickly. It is often used
to check the suitability of a protein for structure determination using NMR, as well as for the optimization of the sample co nditions.
It is one of the standard suite of experiments used for the determination of the solution structure of protein. The HSQC can be
further expanded into three- and four dimensional NMR experiments, such as 15N-TOCSY-HSQC and 15N-NOESY-HSQC (Figure 3.37).
1
H–15N HSQC spectrum of a fragment of an isotopically labeled protein NleG3 -2. Each peak in the spectrum represents a bonded N-H
pair, with its two coordinates corresponding to the chemical shifts of each of the H and N atoms. 1H–15N HSQC spectrum of a
fragment of an isotopically labeled protein NleG3-2. Each peak in the spectrum represents a bonded N-H pair, with its two coordinates
corresponding to the chemical shifts of each of the H and N atoms.
Proteins Page 52
Figure 3.37 1H–15N HSQC spectrum of a fragment of an isotopically labeled protein NleG3-2. Each peak in the spectrum
represents a bonded N-H pair, with its two coordinates corresponding to the chemical shifts of each of the H and N atoms.
Image from:Wu, B., et. al. (2010) PLoS Pathogens 6(6):e1000960.
Cryo-electron Microscopy
Cryogenic-electron microscopy (cryo-EM) has recently emerged as a powerful technique in structural biology that is capable of
delivering high-resolution density maps of macromolecular structures (Fig. 3.38). Resolutions approaching 1.5 Å are now possible and
maps in the 1–4-Å range inform the construction of atomistic models with a high degree of confidence. This new capacity for
investigators to determine macromolecular structures at high resolution and without the need for crystallogenesis has led to an
explosion of interest in adopting cryo-EM.
Protein suspensions are frozen on 3-mm-diameter transmission-electron microscope (TEM) support grids made from a conductive
material (e.g. Cu or Au) that are coated with a carbon film with a regular array of perforations 1 –2 μm in diameter. A total of 3–5 μl of
sample is loaded onto the grid which is then immediately blotted with filter paper with the aim of creating a film of buffer/ protein on
the grid that, when frozen, will be thin enough for the electron beam to penetrate. Optimising the ice thickness is a vital s tep in
sample preparation as thicker layers of ice increase the probability that the incident electron will undergo multiple scatter ing events
and thereby reduce the image quality. In the case of extreme ice thickness, the electron beam does not penetrate at all. Afte r
blotting, the grid is rapidly plunged into a bath of liquid ethane—a very effective cryogen that freezes water with sufficient rapidity
as to prevent formation of ice crystals. The formation of a vitreous layer of ice is the fundamental step in cryo -EM and preserves the
target in a near-native state. The resulting vitreous ice layer with suspended protein molecules must then remain close to liquid
nitrogen temperature (− 196 °C) during storage and imaging in the TEM to prevent phase changes to other types of ice that are not
amenable to high-quality imaging and preservation of protein structure.
Image formation in cryo-EM is primarily by phase contrast, although between 7 and 10% of image contrast is from amplitude contrast.
Amplitude contrast, where an electron is scattered to such an extent it is removed by an aperture or energy filter along the path of
the TEM column, is generally not considered, as the information obtained is usually low resolution compared with phase contra st.
Figure 3.38 Cryo-Electron Microscopy. (a) the Scottish Centrel for Macromolecular Imaging JEOL CryoARM 300. (b) High -resolution
2.2-Å resolution structure of lumazine synthase.
Figure from: Bhella, D. (2019) Biophysical Reviews 11:515-519
Advantages and Disadvantages to Structure Elucidation

Obtaining a pure, highly concentrated (mM) protein sample is a major bottleneck for both x -ray crystallography and NMR. The high
concentration is required because both techniques are insensitive to single molecule analysis, and a large population of a pa rticular
protein is required to overcome the signal-to-noise barrier. On a similar note the sample needs to be very homogenous, so protein
purification is necessary at some point. Cryo-EM requires considerably less protein than for the other two methods, but still ‘a lot’ by
any standard. Typically, Cryo-EM requires preparations at a concentration of 1 mg/ml in a volume of at least 50 μl, whereas
crystallogenesis migh require 500 μlof protein at a concentration of 5 -10 mg/ml. Cryo-Em also requires the protein to be prepared in a
low-salt buffer, with minimal additives, to ensure good freezing and image contrast.
Recombinant protein production using E. coli is the method of choice when large quantities of protein are required. This process
involves taking the gene (often cDNA) of the protein of interest, splicing it into a suitable inducible vector, transforming the vector
into an E. coli host, and growing the culture in a rich medium. The bacterial host will multiply during a growth phase, after which it is
induced to express the protein of interest. If all goes well, the protein will express solubly and in high numbers. Unfortuna tely, this
process is easier saidthan done. Many eukaryotic proteins do not express well in prokaryotic hosts, and oftentimes modificati ons need
Proteins Page 53
process is easier saidthan done. Many eukaryotic proteins do not express well in prokaryotic hosts, and oftentimes modificati ons need
to be made to optimize the bacterial host, codon usage, media, etc. to obtain a decent yield of recombinant protein. Addition ally,
proteins often express insolubly as inclusion bodies and require high concentrations (2M to 8M) of denaturants such as urea o r
guanidine hydrochloride to solubilize them, and then stepwise dialysis into an appropriate buffer to refold them. Alternative ly,
eukaryotic organisms such as S. cerevisiae (yeast), insect and mammalian cell lines can be used, especially when post -translation
modifications are required, though a decrease in yield and increase in overall cost is common with these organisms.
The difficulty of protein production is compounded for NMR by the fact that all proteins need to be 15N and/or 13C labeled, as only
these isotopes have nuclei with + ½ and – ½ spin states which enable the energy transitions required for a radiofrequency NMR signal;
note that 1H also has ½ spin states but is highly abundant.
Protein stability is an issue for both crystallography and NMR. Once a protein has been expressed, purified, and concentrated , it must
maintain its structural integrity for the duration of the experiments. For crystallography, this involves the crystallization process,
where the protein sample is placed in a variety of solutions (most often involving high concentrations of polyethylene glycol ) that
induce crystallization. Often referred to as a voodoo technique, crystallization conditions are tested in a high throughput m ethod using
96-well screening plates, and any hits are further optimized using a larger volume of the particular solution. While a crystalli zation
condition may eventually be found, the process can take anywhere from a few days to even a year or two to happen, making the
crystallization process the rate-limiting step for protein crystallographers. During this time, the protein must stay in solution and
maintain its structure so as to produce a high quality crystal; a condition that is not often the case.
Similarly, a stable, highly concentrated protein sample is required to perform many of the more advanced NMR experiments. Thi s is
because many of these experiments require days and even weeks to run, during which the homogeneity of the solution is key to
acquiring quality spectra. Should the protein unfold or precipitate out of solution during an experiment, the resulting chemi cal change
would either not produce any signal, or one which could not be used for structure/dynamics determination.
For Cryo-EM, working with frozen-hydrated specimens brings a number of challenges both for manipulations and imaging. When
handling cryo-EM grids to load them into the microscope, exposure to atmospheric water vapour rapidly leads to frost buildup on the
grid. Under the TEM, these ice crystals on the grid surface appear as huge boulders that completely block the electron beam. Thus,
grids are kept under liquid nitrogen as much as possible to minimize frost contamination. Problems with ice conditions are common—
insufficient rapid freezing leads to formation of hexagonal ice, while devitrification occurs when samples warm up, leading t o
formation of cubic ice. Various degrees of contamination may occur, and frosting at atmospheric pressure causes the above -mentioned
ice crystal deposition, while contamination within the column or under low-vacuum conditions gives rise to a more subtle artefact.
One of the hallmarks of protein crystallography is that size does not matter. Whether one is working with a 25 kDa monomeric
protein, or a 900 kDa multimeric complex, if it can be crystallized and produce a high -resolution diffraction pattern its structure can
be determined. This is due to the fact that once in crystal form, a protein is in a more -or-less static conformation which, after passing
it through the x-ray beam at different angles, can produce a single structural model. Cryo-EM is similar in this regard. Very large
structures, including massive nucleoprotein complexes, such as the ribosome, can be elucidated using Cryo -EM. The same cannot be
said for NMR.
In NMR, the protein is in a soluble state and therefore in constant movement. The most important movement that governs the sp ectral
quality is that of the molecular tumbling rate. For proteins larger than about 40 kDa, the tumbling rate decreases significan tly, in turn
increasing the transverse relaxation rate (T 2). Essentially, this results in a weaker and rapidly decaying NMR signal, which manifests
itself in peak broadening and spectral overlap.
One of the major advantages of NMR is its ability to record small and large -scale protein dynamics, a phenomenon that is generally
suppressed when a protein is crystallized. Although a crystallized protein may exhibit a certain amount of motion within the lattice,
the motions manifest themselves as static or dynamic disorder, the former of which may result in two different conformations of a
particular region, and the latter in averaged electron density. In general, crystallization may restrict a protein’s natural flexibility and
motions. Cryo-EM suffers from this same limitation as samples are frozen and immobile. However, cryo -EM is capable of capturing a
snap shot of the native structure as freezing is instantaneous and does not require the formation of a crystal lattice.
Crystallography, however, is not left in the cold when it comes to dynamic structure analysis. Time -resolved crystallography can be
used to monitor changes in the protein structure upon addition of some ligand, or change in the environment. Because all prot ein
crystals are highly hydrated, they are able to serve as crucibles for some biochemical reactions. The crystal is typically so aked in a
solution containing the ligand of interest to initiate the biochemical reaction, after which the crystal is quickly placed in to the beam-
line and diffraction pattern is obtained. This can be performed multiple times if necessary to obtain a variety of structural
intermediates. The process though requires many things to go right: the protein cannot become disordered nor should the cryst al
become cracked during the soaking process, and a high-powered synchrotron isrequired to collect high-quality diffraction data over
short exposure times.
In the end, protein x-ray crystallography, cryo-EM and NMR spectroscopy are not mutually exclusive techniques; one can easily pick up
where the other falls short. In analyzing NMR dynamics experiments, for example, one can greatly benefit from existing crysta l
structure data, or cryo-EM data onto which the NMR structural data can be superimposed. Similarly, NMR structure data can be used
to supplement a cryo-EM or crystal structure with more information on the protein’s dynamics, binding information, and conformational
changes in solution.
back to the top

The proteome is the entire set of proteins that is produced or modified by an organism or system. Proteomics has enabled the
identification of ever increasing numbers of protein. This varies with time and distinct requirements, or stresses, that a ce ll or
organism undergoes. Proteomics is an interdisciplinary domain that has benefitted greatly from the genetic information of var ious
genome projects, including the Human Genome Project. It covers the exploration of proteomes from the overall level of protein
composition, structure, and activity. It is an important component of functional genomics.
Proteins Page 54
composition, structure, and activity. It is an important component of functional genomics.
After genomics and transcriptomics, proteomics is the next step in the study of biological systems. It is more complicated th an
genomics because an organism’s genome is more or less constant, whereas proteomes differ from cell to cell and from time to t ime.
Distinct genes are expressed in different cell types, which means that even the basic set of proteins that are produced in a cell needs
to be identified.
In the past this phenomenon was assessed by RNA analysis, but it was found to lack correlation with protein content. Now it i s known
that mRNA is not always translated into protein, and the amount of protein produced for a given amount of mRNA depends on the gene
it is transcribed from and on the current physiological state of the cell. Proteomics confirms the presence of the protein an d provides
a direct measure of the quantity present.
A cell may make different sets of proteins at different times or under different conditions, for example during development, cellular
differentiation, cell cycle, or carcinogenesis. Further increasing proteome complexity, as mentioned, most proteins are able to undergo
a wide range of post-translational modifications.
Therefore, a proteomics study may become complex very quickly, even if the topic of study is restricted. In more ambitious se ttings,
such as when a biomarker for a specific cancer subtype is sought, the proteomics scientist might elect to study multiple bloo d serum
samples from multiple cancer patients to minimise confounding factors and account for experimental noise. Furthermore, many
proteins undergo postranslational modifications such as phosphoyrlation. Many of these post-translational modifications are critical to
the protein’s function. Thus, complicated experimental designs are sometimes necessary to account for the dynamic complexity of the
proteome.
3.6 References
Molnar, C. and Gair, J. (2013) Antibodies. Chapter in Concepts in Biology, Published by B.C. Open Textbook Project. Available at:
https://opentextbc.ca/biology/chapter/23-3-antibodies/
The Human Atlas Project. (2019) Methods. Available at: https://www.proteinatlas.org/learn/method
Uhlén M et al, 2015. Tissue-based map of the human proteome. Science
PubMed:25613900 DOI:10.1126/science.1260419
Thul PJ et al, 2017. A subcellular map of the human proteome. Science.
PubMed:28495876 DOI: 10.1126/science.aal3321
Uhlen M et al, 2017. A pathology atlas of the human cancer transcriptome. Science.
PubMed:28818916 DOI:10.1126/science.aan2507
Ahern, K. and Rajagopal, I. (2019) Biochemistry Free and Easy. Published by Libretexts. Available at:
https://bio.libretexts.org/Bookshelves/Biochemistry/Book%3A_Biochemistry_Free_and_Easy_(Ahern_and_Rajagopal)/09%
3A_Techniques/9.04%3A_Gel_Exclusion_Chromatography.
Magdeldin, S. (2012) Gel Electrophoresis – Principles and Basics. Published by InTech under Creative Commons Attribution 3.0.
Available at: https://pdfs.semanticscholar.org/4b93/70ac3946cec6e12c369679c4178a5ef38e61.pdf
Structural Biochemistry/Proteins/X-ray Crystallography. (2018, November 19). Wikibooks, The Free Textbook Project. Retrieved
15:40, August 17, 2019 from https://en.wikibooks.org/w/index.php?title=Structural_Biochemistry/Proteins/X-
ray_Crystallography&oldid=3488057.
UCD: Biophysics 200A (2019) “NMR Spectroscopy vs X-ray Crystallography”, Chapter published in Current Techniques in Biophysics.
Published by Libretexts and available at: https://phys.libretexts.org/Courses/University_of_California_Davis/UCD%3A_Biophysics_
200A_-_Current_Techniques_in_Biophysics/NMR_Spectroscopy_vs._X-ray_Crystallography
Wikipedia contributors. (2019, June 27). Protein purification. In Wikipedia, The Free Encyclopedia. Retrieved 23:32, July 28, 2019,
from https://en.wikipedia.org/w/index.php?title=Protein_purification&oldid=903657925
Wikipedia contributors. (2019, February 15). Fast protein liquid chromatography. In Wikipedia, The Free Encyclopedia. Retrieved
17:14, August 15, 2019, from https://en.wikipedia.org/w/index.php?title=Fast_protein_liquid_chromatography&oldid=883530035
Wikipedia contributors. (2019, July 9). Protein mass spectrometry. In Wikipedia, The Free Encyclopedia. Retrieved 15:27, August 16,
2019, from https://en.wikipedia.org/w/index.php?title=Protein_mass_spectrometry&oldid=905547289
Wikipedia contributors. (2019, July 8). Peptide synthesis. In Wikipedia, The Free Encyclopedia. Retrieved 06:13, August 17, 2019, from
https://en.wikipedia.org/w/index.php?title=Peptide_synthesis&oldid=905401648
Wikipedia contributors. (2019, July 11). Edman degradation. In Wikipedia, The Free Encyclopedia. Retrieved 06:16, August 17, 2019,
from https://en.wikipedia.org/w/index.php?title=Edman_degradation&oldid=905808326
Bhella, D. (2019) Cryo-electron microscopy: an introduction to the technique, and considerations when working to establish a national
facility. Biochysical Reviews 11: 515-519. Available at: https://link.springer.com/article/10.1007/s12551-019-00571-w
Proteins Page 55
Tuesday, 14 September, 2021 03:54 PM
https://www.masterorganicchemistry.com/2017/05/24/d-and-l-sugars/
• A molecule with n chiral centres has 2n different possible stereoisomers.
Carbohydrates Page 56
CSIR-UGC NET Syllabus
CSIR-UGC National Eligibility Test (NET) for Junior Research Fellowship and Lecturer-ship
LIFE SCIENCES
1. Molecules and their Interaction Relevant to Biology
2. Cellular Organization
3. Fundamental Processes
4. Cell Communication and Cell Signaling
5. Developmental Biology
6. System Physiology – Plant
7. System Physiology – Animal
8. Inheritance Biology
9. Diversity of Life Forms
10. Ecological Principles
11. Evolution and Behavior
12. Applied Biology
13. Methods in Biology
For note making:::

WHAT
Uniqueness
How
When
Mode of action
Mutation cases
Connectivity
Specific Naming (if any)
Relation
1. MOLECULES AND THEIR INTERACTION RELAVENT TO BIOLOGY

A. Structure of atoms, molecules and chemical bonds.
B Composition, structure and function of biomolecules (carbohydrates, lipids, proteins, nucleic acids and vitamins).
C. Stablizing interactions (Van der Waals, electrostatic, hydrogen bonding, hydrophobic interaction, etc.).
D Principles of biophysical chemistry (pH, buffer, reaction kinetics, thermodynamics, colligative properties).
E. Bioenergetics, glycolysis, oxidative phosphorylation, coupled reaction, group transfer, biological energy transducers.
F. Principles of catalysis, enzymes and enzyme kinetics, enzyme regulation, mechanism of enzyme catalysis, isozymes
G. Conformation of proteins (Ramachandran plot, secondary structure, domains, motif and folds).
H. Conformation of nucleic acids (helix (A, B, Z), t-RNA, micro-RNA).
I. Stability of proteins and nucleic acids.
J. Metabolism of carbohydrates, lipids, amino acids nucleotides and vitamins.
2. CELLULAR ORGANIZATION
A) Membrane structure and function

(Structure of model membrane, lipid bilayer and membrane protein diffusion, osmosis, ion channels, active transport, membrane
pumps, mechanism of sorting and regulation of intracellular transport,electrical properties of membranes).
B) Structural organization and function of intracellular organelles (Cell wall, nucleus, mitochondria, Golgi bodies, lysosomes,
endoplasmic reticulum, peroxisomes, plastids, vacuoles, chloroplast, structure & function of cytoskeleton and its role in motility).
C) Organization of genes and chromosomes (Operon, unique and repetitive DNA, interrupted genes, gene families, structure of
chromatin and chromosomes, heterochromatin, euchromatin, transposons).
D) Cell division and cell cycle (Mitosis and meiosis, their regulation, steps in cell cycle, regulation and control of cell cycle).
E) Microbial Physiology (Growth yield and characteristics, strategies of cell division, stress response)
3. FUNDAMENTAL PROCESSES
A) DNA replication, repair and recombination (Unit of replication, enzymes involved, replication origin and replication fork, fidelity of
replication, extrachromosomal replicons, DNA damage and repair mechanisms, homologous and site-specific recombination).
B) RNA synthesis and processing (transcription factors and machinery, formation of initiation complex, transcription activator and
repressor, RNA polymerases, capping, elongation, and termination, RNA processing, RNA editing, splicing, and polyadenylation,
Details Page 57
repressor, RNA polymerases, capping, elongation, and termination, RNA processing, RNA editing, splicing, and polyadenylation,
structure and function of different types of RNA, RNA transport).
C) Protein synthesis and processing (Ribosome, formation of initiation complex, initiation factors and their regulation, elongation and
elongation factors, termination, genetic code, aminoacylation of tRNA, tRNA-identity, aminoacyl tRNA synthetase, and translational
proof-reading, translational inhibitors, Post- translational modification of proteins).
D) Control of gene expression at transcription and translation level (regulating the expression of phages, viruses, prokaryotic and
eukaryotic genes, role of chromatin in gene expression and gene silencing).
4. Cell communication and cell signaling
A) Host parasite interaction Recognition and entry processes of different pathogens like bacteria, viruses into animal and plant host
cells, alteration of host cell behavior by pathogens, virus-induced cell transformation, pathogen-induced diseases in animals and plants,
cell-cell fusion in both normal and abnormal cells.
B) Cell signaling Hormones and their receptors, cell surface receptor, signaling through G-protein coupled receptors, signal transduction
pathways, second messengers, regulation of signaling pathways, bacterial and plant twocomponent systems, light signaling in plants,
bacterial chemotaxis and quorum
sensing.
C) Cellular communication Regulation of hematopoiesis, general principles of cell communication, cell adhesion and roles of different
adhesion molecules, gap junctions, extracellular matrix, integrins, neurotransmission and its regulation.
D) Cancer
Genetic rearrangements in progenitor cells, oncogenes, tumor suppressor genes, cancer and the cell cycle, virus-induced cancer,
metastasis, interaction of cancer cells with normal cells, apoptosis, therapeutic interventions of uncontrolled cell
growth.
E) Innate and adaptive immune system Cells and molecules involved in innate and adaptive immunity, antigens, antigenicity and
immunogenicity. B and T cell epitopes, structure and function of antibody molecules. generation of antibody
diversity, monoclonal antibodies, antibody engineering, antigen-antibody interactions, MHC molecules, antigen processing and
presentation, activationand differentiation of B and T cells, B and T cell receptors, humoral and cellmediated immune responses,
primary and secondary immune modulation, the complement system, Toll-like receptors, cell-mediated effector functions,
inflammation, hypersensitivity and autoimmunity, immune response during bacterial (tuberculosis), parasitic (malaria) and viral (HIV)
infections, congenital and acquired immunodeficiencies, vaccines.
5. DEVELOPMENTAL BIOLOGY
A) Basic concepts of development : Potency, commitment, specification, induction, competence, determination and differentiation;
morphogenetic gradients; cell fate and cell lineages; stem cells; genomic equivalence and the cytoplasmic determinants; imprinting;
mutants and transgenics in analysis of development
B) Gametogenesis, fertilization and early development: Production of gametes, cell surface molecules in sperm-egg recognition in
animals; embryo sac development and double fertilization in plants; zygote formation, cleavage, blastula formation, embryonic fields,
gastrulation and formation of germ layers in animals; embryogenesis, establishment of symmetry in plants; seed formation and
germination.
C) Morphogenesis and organogenesis in animals : Cell aggregation and differentiation in Dictyostelium; axes and pattern formation in
Drosophila, amphibia and chick; organogenesis – vulva formation in Caenorhabditis elegans, eye lens induction, limb development and
regeneration in vertebrates; differentiation of neurons, post embryonic development- larval formation, metamorphosis; environmental
regulation of normal development; sex determination.
D) Morphogenesis and organogenesis in plants: Organization of shoot and root apical meristem; shoot and root development; leaf
development and phyllotaxy; transition to flowering, floral meristems and floral development in Arabidopsis and Antirrhinum
E) Programmed cell death, aging and senescence
6. SYSTEM PHYSIOLOGY - PLANT
A. Photosynthesis - Light harvesting complexes; mechanisms of electron transport; photoprotective mechanisms; CO2 fixation-C3, C4
and CAM pathways.
B. Respiration and photorespiration – Citric acid cycle; plant mitochondrial electron transport and ATP synthesis; alternate oxidase;
photorespiratory pathway.
C. Nitrogen metabolism - Nitrate and ammonium assimilation; amino acid biosynthesis.
D. Plant hormones – Biosynthesis, storage, breakdown and transport; physiological effects and mechanisms of action.
E. Sensory photobiology - Structure, function and mechanisms of action of phytochromes, cryptochromes and phototropins; stomatal
movement; photoperiodism and biological clocks.
F. Solute transport and photoassimilate translocation – uptake, transport and translocation of water, ions, solutes and macromolecules
from soil, through cells, across membranes, through xylem and phloem; transpiration; mechanisms of
loading and unloading of photoassimilates.
G. Secondary metabolites - Biosynthesis of terpenes, phenols and nitrogenous compounds and their roles.
H. Stress physiology – Responses of plants to biotic (pathogen and insects) and abiotic (water, temperature and salt) stresses.
7. SYSTEM PHYSIOLOGY - ANIMAL
Details Page 58
A. Blood and circulation - Blood corpuscles, haemopoiesis and formed elements, plasma function, blood volume, blood volume
regulation, blood groups, haemoglobin, immunity, haemostasis.
B. Cardiovascular System: Comparative anatomy of heart structure, myogenic heart, specialized tissue, ECG – its principle and
significance, cardiac cycle, heart as a pump, blood pressure, neural and chemical regulation of all above.
C. Respiratory system - Comparison of respiration in different species, anatomical considerations, transport of gases, exchange of gases,
waste elimination, neural and chemical regulation of respiration.
D. Nervous system - Neurons, action potential, gross neuroanatomy of the brain and spinal cord, central and peripheral nervous
system, neural control of muscle tone and posture.
E. Sense organs - Vision, hearing and tactile response.
F. Excretory system - Comparative physiology of excretion, kidney, urine formation, urine concentration, waste elimination, micturition,
regulation of water balance, blood volume, blood pressure, electrolyte balance, acid-base balance.
G. Thermoregulation - Comfort zone, body temperature – physical, chemical, neural regulation, acclimatization.
H. Stress and adaptation
I. Digestive system - Digestion, absorption, energy balance, BMR.
J. Endocrinology and reproduction - Endocrine glands, basic mechanism of hormone action, hormones and diseases; reproductive
processes, gametogenesis, ovulation, neuroendocrine regulation
8. INHERITANCE BIOLOGY
A) Mendelian principles : Dominance, segregation, independent assortment.

B) Concept of gene : Allele, multiple alleles, pseudoallele, complementation tests
C) Extensions of Mendelian principles : Codominance, incomplete dominance, gene interactions, pleiotropy, genomic imprinting,
penetrance and expressivity, phenocopy, linkage and crossing over, sex linkage, sex limited and sex influenced characters.
D) Gene mapping methods : Linkage maps, tetrad analysis, mapping with molecular markers, mapping by using somatic cell hybrids,
development of mapping population in plants.
E) Extra chromosomal inheritance : Inheritance of Mitochondrial and chloroplast genes, maternal inheritance.
F) Microbial genetics : Methods of genetic transfers – transformation, conjugation, transduction and sex-duction, mapping genes by
interrupted mating, fine structure analysis of genes.
G) Human genetics : Pedigree analysis, lod score for linkage testing, karyotypes, genetic disorders.
H) Quantitative genetics : Polygenic inheritance, heritability and its measurements, QTL mapping.
I) Mutation : Types, causes and detection, mutant types – lethal, conditional, biochemical, loss of function, gain of function, germinal
verses somatic mutants, insertional mutagenesis.
J) Structural and numerical alterations of chromosomes : Deletion, duplication, inversion, translocation, ploidy and their genetic
implications.
K) Recombination : Homologous and non-homologous recombination including transposition.
9. DIVERSITY OF LIFE FORMS:
A. Principles & methods of taxonomy:

Concepts of species and hierarchical taxa, biological nomenclature, classical & quantititative methods of taxonomy of plants, animals
and microorganisms.
B. Levels of structural organization:
Unicellular, colonial and multicellular forms. Levels of organization of tissues, organs & systems. Comparative anatomy, adaptive
radiation, adaptive modifications.
C. Outline classification of plants, animals & microorganisms:
Important criteria used for classification in each taxon. Classification of plants, animals and microorganisms. Evolutionary relationships
among taxa.
D. Natural history of Indian subcontinent:
Major habitat types of the subcontinent, geographic origins and migrations of species. Comman Indian mammals, birds. Seasonality and
phenology of the subcontinent.
E. Organisms of health & agricultural importance:
Common parasites and pathogens of humans, domestic animals and crops.
F. Organisms of conservation concern:
Rare, endangered species. Conservation strategies.
10. ECOLOGICAL PRINCIPLES
The Environment: Physical environment; biotic environment; biotic and abiotic interactions.
Habitat and Niche: Concept of habitat and niche; niche width and overlap; fundamental and realized niche; resource partitioning;
character displacement.
Population Ecology: Characteristics of a population; population growth curves; population regulation; life history strategies (r and K
selection); concept of metapopulation – demes and dispersal, interdemic extinctions, age structured
populations.
Species Interactions: Types of interactions, interspecific competition, herbivory, carnivory, pollination, symbiosis.
Community Ecology: Nature of communities; community structure and attributes; levels of species diversity and its measurement;
Details Page 59
Community Ecology: Nature of communities; community structure and attributes; levels of species diversity and its measurement;
edges and ecotones.
Ecological Succession: Types; mechanisms; changes involved in succession; concept of climax.
Ecosystem Ecology: Ecosystem structure; ecosystem function; energy flow and mineral cycling (C,N,P); primary production and
decomposition; structure and function of some Indian ecosystems: terrestrial (forest, grassland) and aquatic (fresh water,
marine, eustarine).
Biogeography: Major terrestrial biomes; theory of island biogeography; biogeographical zones of India.
Applied Ecology: Environmental pollution; global environmental change; biodiversity: status, monitoring and documentation; major
drivers of biodiversity change; biodiversity management approaches.
Conservation Biology: Principles of conservation, major approaches to management, Indian case studies on conservation/management
strategy (Project Tiger, Biosphere reserves).
11. EVOLUTION AND BEHAVIOUR
A. Emergence of evolutionary thoughts

Lamarck; Darwin–concepts of variation, adaptation, struggle, fitness and natural selection; Mendelism; Spontaneity of mutations; The
evolutionary synthesis.
B. Origin of cells and unicellular evolution:
Origin of basic biological molecules; Abiotic synthesis of organic monomers and polymers; Concept of Oparin and Haldane; Experiment
of Miller (1953); The first cell; Evolution of prokaryotes; Origin of eukaryotic cells; Evolution of unicellular eukaryotes; Anaerobic
metabolism, photosynthesis and aerobic metabolism.
C. Paleontology and Evolutionary History:
The evolutionary time scale; Eras, periods and epoch; Major events in the evolutionary time scale; Origins of unicellular and multi
cellular organisms; Major groups of plants and animals; Stages in primate evolution including Homo.
D. Molecular Evolution:
Concepts of neutral evolution, molecular divergence and molecular clocks; Molecular tools in phylogeny, classification and
identification; Protein and nucleotide sequence analysis; origin of new genes and proteins; Gene duplication and divergence.
E. The Mechanisms:
Population genetics – Populations, Gene pool, Gene frequency; Hardy-Weinberg Law; concepts and rate of change in gene frequency
through natural selection, migration and random genetic drift; Adaptive radiation; Isolating mechanisms; Speciation; Allopatricity
and Sympatricity; Convergent evolution; Sexual selection; Co-evolution.
F. Brain, Behavior and Evolution:
Approaches and methods in study of behavior; Proximate and ultimate causation; Altruism and evolution-Group selection, Kin selection,
Reciprocal altruism; Neural basis of learning, memory, cognition, sleep and arousal; Biological clocks; Development of behavior; Social
communication; Social dominance; Use of space and territoriality; Mating systems, Parental investment and Reproductive success;
Parental care; Aggressive behavior; Habitat selection and optimality in foraging; Migration, orientation and navigation; Domestication
and behavioral changes.
12. APPLIED BIOLOGY:

A. Microbial fermentation and production of small and macro molecules.
B. Application of immunological principles, vaccines, diagnostics. Tissue and cell culture methods for plants and animals.
C. Transgenic animals and plants, molecular approaches to diagnosis and strain identification.
D. Genomics and its application to health and agriculture, including gene therapy.
E. Bioresource and uses of biodiversity.
F. Breeding in plants and animals, including marker – assisted selection
G. Bioremediation and phytoremediation
H. Biosensors
13. METHODS IN BIOLOGY

A. Molecular Biology and Recombinant DNA methods:
Isolation and purification of RNA , DNA (genomic and plasmid) and proteins, different separation methods.
Analysis of RNA, DNA and proteins by one and two dimensional gel electrophoresis, Isoelectric focusing gels.
Molecular cloning of DNA or RNA fragments in bacterial and eukaryotic systems.
Expression of recombinant proteins using bacterial, animal and plant vectors.
Isolation of specific nucleic acid sequences
Generation of genomic and cDNA libraries in plasmid, phage, cosmid, BAC and YAC vectors.
In vitro mutagenesis and deletion techniques, gene knock out in bacterial and eukaryotic organisms.
Protein sequencing methods, detection of post translation modification of proteins.
DNA sequencing methods, strategies for genome sequencing.
Methods for analysis of gene expression at RNA and protein level, large scale expression, such as micro array based techniques
Isolation, separation and analysis of carbohydrate and lipid molecules
RFLP, RAPD and AFLP techniques
B. Histochemical and Immunotechniques

Antibody generation, Detection of molecules using ELISA, RIA, western blot, immunoprecipitation, fluocytometry and
immunofluorescence microscopy, detection of molecules in living cells, in situ localization by techniques such as FISH
Details Page 60
immunofluorescence microscopy, detection of molecules in living cells, in situ localization by techniques such as FISH
and GISH.
C Biophysical Method:
Molecular analysis using UV/visible, fluorescence, circular dichroism, NMR and ESR spectroscopy Molecular structure determination
using X-ray diffraction and NMR, Molecular analysis using light scattering, different types of mass spectrometry and surface plasma
resonance methods.
D Statisitcal Methods:
Measures of central tendency and dispersal; probability distributions (Binomial, Poisson and normal); Sampling distribution; Difference
between parametric and non-parametric statistics; Confidence Interval; Errors; Levels of significance; Regression and Correlation; t-
test; Analysis of variance; X2 test;; Basic introduction to Muetrovariate statistics, etc.
E. Radiolabeling techniques:
Detection and measurement of different types of radioisotopes normally used in biology, incorporation of radioisotopes in biological
tissues and cells, molecular imaging of radioactive material, safety guidelines.
F. Microscopic techniques:
Visualization of cells and subcellular components by light microscopy, resolving powers of different microscopes, microscopy of living
cells, scanning and transmission microscopes, different fixation and staining techniques for EM, freeze-etch and freeze fracture methods
for EM, image processing methods in microscopy.
G. Electrophysiological methods:
Single neuron recording, patch-clamp recording, ECG, Brain activity recording, lesion and stimulation of brain, pharmacological testing,
PET, MRI, fMRI, CAT .
H. Methods in field biology:

Methods of estimating population density of animals and plants, ranging patterns through direct, indirect and remote observations,
sampling methods in the study of behaviour, habitat characterization: ground and remote sensing methods.
Details Page 61
Five Kingdom Classification
Saturday, 20 November, 2021 10:46 PM
Biological Classification
• Biological classification of plants and animals was first proposed by Aristotle on the basis of simple morphological characters.
• Linnaeus later classified all living organisms into two kingdoms – Plantae and Animalia.
• Whittaker proposed an elaborate five kingdom classification – Monera, Protista, Fungi, Plantae and Animalia.
• The main criteria of the five kingdom classification were cell structure, body organisation, mode of nutrition and reproduction, and phylogenetic
relationships [evolutionary development and diversification of a species].
At present, the biological classification includes:

1. Kingdom Monera
2. Kingdom Protista
3. Kingdom Fungi
4. Kingdom Plantae
5. Kingdom Animalia
6. Viruses, Viroids and Lichens
• In the five kingdom classification, bacteria are included in Kingdom Monera.
• Kingdom Protista includes all single-celled eukaryotes such as Chrysophytes, Dinoflagellates, Euglenoids, Slime-moulds and Protozoans.
• Members of Kingdom Fungi show a great diversity in structures and habitat. Most fungi are saprophytic in their mode of nutrition.
• The plantae includes all eukaryotic chlorophyll-containing organisms. Algae, bryophytes, pteridophytes, gymnosperms and angiosperms are
included in this group.
• The heterotrophic eukaryotic, multicellular organisms lacking a cell wall are included in the Kingdom Animalia.
• Some acellular organisms like viruses and viroids as well as the lichens are not included in the five kingdom system of classification.
Animal Classification Page 62

Kingdom Monera
• The organisms in this group are
1. prokaryotes == do not have a defined nucleus or organelles {Prokaryotic Cells vs. Eukaryotic Cells}.
2. unicellular == do any of them show multi-cellular body designs.
• This group includes all Some well-known bacteria include blue-green algae or cyanobacteria [have cell walls], and mycoplasma [doesn’t possess
a Cell Wall].
• They are the most abundant micro-organisms and live in extreme habitats.
• Some of them have cell walls [bacteria] while some do not [mycoplasma].
• The mode of nutrition of organisms in this group can be either by synthesizing their own food (autotrophic) or getting it from the environment
(heterotrophic). Many of them live in or on other organisms as parasites.
• Bacteria are grouped under four categories based on their shape
1. the spherical Coccus
2. the rod-shaped Bacillus
3. the comma-shaped Vibrium
4. the spiral Spirillum
• Some of the bacteria are autotrophic, i.e., they synthesise their own food. They may be photosynthetic autotrophic or chemosynthetic
autotrophic (metabolic synthesis of organic compounds by living organisms using energy derived from reactions involving inorganic chemicals).
Archaebacteria
• These bacteria are special since they live in some of the most harsh habitats such as
• extreme salty areas (halophiles),
• hot springs (thermoacidophiles) and
• marshy areas (methanogens) {Microbes In Human Welfare | Useful Microbes}.
• Archaebacteria differ from other bacteria in having a different cell wall structure and this feature is responsible for their survival in extreme
conditions.
• Methanogens are present in the gut of several ruminant animals such as cows and buffaloes and they are responsible for the production of
methane (biogas) from the dung of these animals.
Eubacteria
• There are thousands of different eubacteria or ‘true bacteria’.
• They are characterized by the presence of a rigid cell wall, and if motile, a flagellum.
Photosynthetic bacteria
• The cyanobacteria (also referred to as blue-green algae) have chlorophyll a similar to green plants and are photosynthetic autotrophs.
• The cyanobacteria are unicellular, colonial, filamentous, freshwater/marine or terrestrial algae. The colonies are generally surrounded by
gelatinous sheath.
• They often form blooms [algal blooms] in polluted water bodies.
• Some of these organisms can fix atmospheric nitrogen in specialized cells called heterocysts, e.g., Nostoc and Anabaena.
Chemosynthetic bacteria
• Chemosynthetic autotrophic bacteria oxidise various inorganic substances such as nitrates, nitrites and ammonia and use the released energy
for their ATP production.
• They play a great role in recycling nutrients like nitrogen, phosphorous, iron and sulphur.
Heterotrophic bacteria
• Heterotrophic bacteria are the most abundant in nature. The majority are important decomposers.
• Many of them have a significant impact on human affairs. They are helpful in making curd from milk, production of antibiotics, fixing nitrogen
in legume roots, etc {Microbes In Human Welfare | Useful Microbes}.
• Some are pathogens causing damage to human beings, crops, farm animals and pets.
• Cholera, typhoid, tetanus, citrus canker are well known diseases caused by different bacteria {Diseases Caused by Microorganisms, Diseases |
Acute, Chronic, Communicable Diseases}.
Reproduction
• Bacteria reproduce mainly by fission.
• Sometimes, under unfavorable conditions, they produce spores.
• They also reproduce by a sort of sexual reproduction by adopting a primitive type of DNA transfer from one bacterium to the other.
Mycoplasma
• The Mycoplasma are organisms that completely lack a cell wall.
• They are the smallest living cells known and can survive without oxygen.

• They are the smallest living cells known and can survive without oxygen.
• Many mycoplasma are pathogenic in animals and plants.
Kingdom Protista
• All single-celled eukaryotes are placed under Protista [Prokaryotic Cells vs. Eukaryotic Cells].
• Boundaries of this kingdom are not well defined. This kingdom forms a link with the others dealing with plants, animals and fungi.
• In this group we include Chrysophytes, Dinoflagellates, Euglenoids, Slime moulds and Protozoans. Examples are unicellular algae, diatoms and
protozoans.
• Their mode of nutrition can be autotrophic or heterotrophic.
• Members of Protista are primarily aquatic. Some have flagella or cilia that helps in movement.
• Protists reproduce asexually and sexually by a process involving cell fusion and zygote formation.
Chrysophytes
• This group includes diatoms and golden algae (desmids).
• Most of them are photosynthetic. Diatoms are the chief ‘producers’ in the oceans.
• They are found in fresh water as well as in marine environments. They are microscopic and float passively in water currents (plankton).
• In diatoms the cell walls form two thin overlapping shells. The walls are embedded with silica and thus the walls are indestructible. Thus,
diatoms have left behind large amount of cell wall deposits in their habitat; this accumulation over billions of years is referred to as
‘diatomaceous earth’. Being gritty this soil is used in polishing, filtration of oils and syrups.
Dinoflagellates
• These organisms are mostly marine and photosynthetic.
• They appear yellow, green, brown, blue or red depending on the main pigments present in their cells.
• The cell wall has stiff cellulose plates on the outer surface.
• Most of them have two flagella; one lies longitudinally and the other transversely in a furrow between the wall plates.
• Very often, red dinoflagellates (Example: Gonyaulax) undergo such rapid multiplication that they make the sea appear red (red tides).
• Toxins released by such large numbers may even kill other marine animals such as fishes.
Euglenoids
• Majority of them are fresh water organisms found in stagnant water.
• Instead of a cell wall, they have a protein rich layer called pellicle which makes their body flexible.
• They have two flagella, a short and a long one.
• Though they are photosynthetic in the presence of sunlight, when deprived of sunlight they behave like heterotrophs by predating on other
smaller organisms.
• Interestingly, the pigments of euglenoids are identical to those present in higher plants. Example: Euglena.
Slime Moulds
• Slime moulds are saprophytic protists.
• The body moves along decaying twigs and leaves engulfing organic material.
• Under suitable conditions, they form an aggregation called plasmodium which may grow and spread over several feet.
• During unfavorable conditions, the plasmodium differentiates and forms fruiting bodies bearing spores at their tips. The spores possess true
walls. They are extremely resistant and survive for many years, even under adverse conditions. The spores are dispersed by air currents.
Protozoans
• All protozoans are heterotrophs and live as predators or parasites. They are believed to be primitive relatives of animals. There are four major
groups of protozoans.
Amoeboid protozoans
• These organisms live in fresh water, sea water or moist soil.
• They move and capture their prey by putting out pseudopodia (false feet) as in Amoeba.
• Marine forms have silica shells on their surface. Some of them such as Entamoeba are parasites.
Flagellated protozoans
• The members of this group are either free-living or parasitic. They have flagella.
• The parasitic forms cause diseases such as sleeping sickness. Example: Trypanosoma.
Ciliated protozoans
• These are aquatic, actively moving organisms because of the presence of thousands of cilia.
• They have a cavity (gullet) that opens to the outside of the cell surface. The coordinated movement of rows of cilia causes the water laden with
food to be steered into the gullet. Example: Paramoecium.
Sporozoans
• This includes diverse organisms that have an infectious spore-like stage in their life cycle.
• The most notorious is Plasmodium (malarial parasite) which causes malaria, a disease which has a staggering effect on human population
{Diseases Caused by Microorganisms}.

Kingdom Fungi
• These are heterotrophic eukaryotic organisms.
• Most fungi are heterotrophic and absorb soluble organic matter from dead substrates and hence are called saprophytes.
• Those that depend on living plants and animals are called parasites.
• Some fungal species live in permanent mutually dependent relationships with bluegreen algae (or cyanobacteria). Such relationships are called
symbiotic. These symbiobic life forms are called lichens. They can also live as symbionts in association with roots of higher plants as
mycorrhiza.
• Fungi + Bluegreen algae (Cyanobacteria) == Lichens. (Prelims 2014)
Q1. Lichens, which are capable of initiating ecological succession even on a bare rock, are actually a symbiotic association of
1. algae and bacteria
2. algae and fungi
3. bacteria and fungi
4. fungi and mosses
• Reproduction in fungi can take place by vegetative means – fragmentation, fission and budding.
• Asexual reproduction is by spores called conidia or sporangiospores or zoospores, and sexual reproduction is by oospores, ascospores and
basidiospores.
• The various spores are produced in distinct structures called fruiting bodies. The sexual cycle involves the following three steps:
1. Fusion of protoplasms between two motile or non-motile gametes called plasmogamy.
2. Fusion of two nuclei called karyogamy.
3. Meiosis in zygote resulting in haploid spores {Meiosis | Mitosis – Meiosis Comparison}.
• When a fungus reproduces sexually, two haploid hyphae of compatible mating types come together and fuse. In some fungi the fusion of two
haploid cells immediately results in diploid cells (2n).
• However, in other fungi (ascomycetes and basidiomycetes), an intervening dikaryotic stage (n + n, i.e., two nuclei per cell) occurs; such a
condition is called a dikaryon and the phase is called dikaryophase of fungus. Later, the parental nuclei fuse and the cells become diploid. The
fungi form fruiting bodies in which reduction division occurs, leading to formation of haploid spores.
• Many of fungi have the capacity to become multicellular organisms at certain stages in their lives.
• They have cell-walls made of a tough complex sugar called chitin.
• Fungi are cosmopolitan and occur in air, water, soil and on animals and plants.
• They prefer to grow in warm and humid places. With the exception of yeasts which are unicellular, fungi are filamentous.
• Their bodies consist of long, slender thread-like structures called hyphae. The network of hyphae is known as mycelium.
• Some hyphae are continuous tubes filled with multinucleated cytoplasm – these are called coenocytic hyphae.
• Others have septae or cross walls in their hyphae. The cell walls of fungi are composed of chitin and polysaccharides {Carbohydrates |
Monosaccharides | Polysaccharides}.
• When your bread develops a mould or your orange rots it is because of fungi.
• The common mushroom you eat and toadstools are also fungi.
• White spots seen on mustard leaves are due to a parasitic fungus.
• Some unicellular fungi, e.g., yeast are used to make bread and beer.
• Other fungi cause diseases in plants and animals; wheat rust-causing Puccinia is an important example.
• Some are the source of antibiotics, e.g., Penicillium.
Phycomycetes
• Asexual reproduction takes place by zoospores (motile) or by aplanospores (non-motile).
• These spores are endogenously produced in sporangium.
• A zygospore is formed by fusion of two gametes.
• These gametes are similar in morphology (isogamous) or dissimilar (anisogamous or oogamous).
• Some common examples are Mucor, Rhizopus (the bread mould mentioned earlier) and Albugo (the parasitic fungi on mustard).
Ascomycetes
• Commonly known as sac-fungi, the as comycetes are mostly multicellular, e.g., Penicillium, or rarely unicellular, e.g., yeast (Saccharomyces).
Basidiomycetes
• Commonly known forms of basidiomycetes are mushrooms, bracket fungi or puffballs.
• They grow in soil, on logs and tree stumps and in living plant bodies as parasites, e.g., rusts and smuts.
• The asexual spores are generally not found, but vegetative reproduction by fragmentation is common.
• The sex organs are absent, but plasmogamy is brought about by fusion of two vegetative or somatic cells of different strains or genotypes. The
resultant structure is dikaryotic.
Deuteromycetes
Deuteromycetes
• Commonly known as imperfect fungi because only the asexual or vegetative phases of these fungi are known.
Kingdom Plantae
• These are multicellular eukaryotes with cell walls mainly made of cellulose {Plant Cell vs. Animal Cell}.
• They are autotrophs and use chlorophyll for photosynthesis.
• A few members are partially heterotrophic such as the insectivorous plants or parasites. Bladderwort and Venus fly trap are examples of
insectivorous plants and Cuscuta is a parasite.
• Plantae includes algae, bryophytes, pteridophytes, gymnosperms and angiosperms.
• Life cycle of plants has two distinct phases – the diploid sporophytic and the haploid gametophytic – that alternate with each other.
• The lengths of the haploid and diploid phases, and whether these phases are free-living or dependent on others, vary among different groups in
plants. This phenomenon is called alternation of generation.
Kingdom Animalia
• These include all organisms which are multicellular eukaryotes without cell walls. They are heterotrophs.
• They directly or indirectly depend on plants for food. They digest their food in an internal cavity and store food reserves as glycogen or fat
{ Carbohydrates , Fats – Healthy Fats and Unhealthy Fats}.
• Their mode of nutrition is holozoic – by ingestion of food.
• They follow a definite growth pattern and grow into adults that have a definite shape and size.
• Higher forms show elaborate sensory and neuromotor mechanism. Most of them are capable of locomotion.
• The sexual reproduction is by copulation of male and female followed by embryological development.
Viruses, Viroids and Lichens

• In the five kingdom classification of Whittaker {Biological Classification} there is no mention of some acellular organisms like viruses and viroids,
and lichens. These are briefly introduced here.
• Viruses did not find a place in classification since they are not truly ‘living’, if we understand living as those organisms that have a cell structure.
• The viruses are non-cellular organisms that are characterized by having an inert crystalline structure outside the living cell.
• Viruses are obligate parasites. Once they infect a cell they take over the machinery of the host cell to replicate themselves, killing the host.
• The name virus that means venom or poisonous fluid was given by Pasteur.
• In addition to proteins, viruses also contain genetic material, that could be either RNA or DNA. No virus contains both RNA and DNA.
• In general, viruses that infect plants have single stranded RNA and viruses that infect animals have either single or double stranded RNA or
double stranded DNA.
• Bacterial viruses or bacteriophages (viruses that infect the bacteria) are usually double stranded DNA viruses
• The protein coat called capsid made of small subunits called capsomeres, protects the nucleic acid. These capsomeres are arranged in helical or
polyhedral geometric forms.
• Viruses cause diseases like mumps, small pox, herpes and influenza. AIDS in humans is also caused by a virus.
• In plants, the symptoms can be mosaic formation, leaf rolling and curling, yellowing and vein clearing, dwarfing and stunted growth.
Viroids
• Viroids are infectious agents that are smaller than viruses. A viroid was found to be a free RNA; it lacked the protein coat that is found in
viruses, hence the name viroid. The RNA of the viroid was of low molecular weight. Viroids caused potato spindle tuber disease.
Lichens
• Lichens are symbiotic associations i.e. mutually useful associations, between algae and fungi.
• The algal component is known as phycobiont and fungal component as mycobiont, which are autotrophic and heterotrophic, respectively.
• Algae prepare food for fungi and fungi provide shelter and absorb mineral nutrients and water for its partner.
• So close is their association that if one saw a lichen in nature one would never imagine that they had two different organisms within them.
• Lichens are very good pollution indicators – they do not grow in polluted areas.
From <https://www.pmfias.com/five-kingdom-classification-plants-animals/>

Animal Kingdom
Friday, 29 October, 2021 12:02 AM
Basis for Animal Kingdom Classification

Classification of Animal Kingdom is based on various fundamental features like –
1. Levels of Organisation,
2. Symmetry,
3. Diploblastic and Triploblastic Organisation,
4. Coelom development,
5. Segmentation of the body and
6. Presense or absence of Notochord.
• The broad classification of Animalia based on common fundamental features:
Levels of Organisation
• Though all members of Animalia are multicellular, all of them do not exhibit the same pattern
of organisation of cells.
• For example, in sponges, the cells are arranged as loose cell aggregates, i.e., they exhibit
cellular level of organisation. Some division of labour (activities) occur among the cells.
• In coelenterates, the arrangement of cells is more complex. Here the cells performing the
same function are arranged into tissues, hence is called tissue level of organisation.
• A still higher level of organisation, i.e., organ level [organ level of organisation] is exhibited by
members of Platyhelminthes and other higher phyla where tissues are grouped together to
form organs, each specialised for a particular function.
• In animals like Annelids, Arthropods, Molluscs, Echinoderms and Chordates, organs have
associated to form functional systems, each system concerned with a specific physiological
function. This pattern is called organ system level of organisation.
• Organ systems in different groups of animals exhibit various patterns of complexities.
• For example, the digestive system in Platyhelminthes (incomplete digestive system) has only
a single opening to the outside of the body that serves as both mouth and anus, and is hence
called incomplete. A complete digestive system has two openings, mouth and anus.
• Similarly, the circulatory system may be of two types: open type in which the blood is pumped
out of the heart and the cells and tissues are directly bathed in it and closed type in which the
blood is circulated through a series of vessels of varying diameters (arteries, veins and
capillaries).
Symmetry
• Animals can be categorised on the basis of their symmetry.
• Sponges are mostly asymmetrical, i.e., any plane that passes through the centre does not
divide them into equal halves.
• When any plane passing through the central axis of the body divides the organism into two
identical halves, it is called radial symmetry. Coelenterates, Ctenophores and Echinoderms
have this kind of body plan.
• Animals like Annelids, Arthropods, etc., where the body can be divided into identical left and
right halves in only one plane, exhibit bilateral symmetry.

Diploblastic and Triploblastic Organisation
• Animals in which the cells are arranged in two embryonic layers, an external ectoderm and an
internal endoderm, are called diploblastic animals, e.g., Coelenterates. An undifferentiated
layer, mesoglea, is present in between the ectoderm and the endoderm.
• Those animals in which the developing embryo has a third germinal layer, mesoderm, in
between the ectoderm and endoderm, are called triploblastic animals (platyhelminthes to
chordates).
Figure: Showing germinal layers : (a) Diploblastic (b) Triploblastic

Coelom
• Presence or absence of a cavity between the body wall and the gut wall is very important in
classification.
• The body cavity, which is lined by mesoderm is called coelom.
• Animals possessing coelom are called coelomates, e.g., Annelids, Molluscs, Arthropods,
Echinoderms, Hemichordates & Chordates.
• In some animals, the body cavity is not lined by mesoderm, instead, the mesoderm is present
as scattered pouches in between the ectoderm and endoderm. Such a body cavity is called
pseudocoelom and the animals possessing them are called pseudocoelomates, e.g.,
Aschelminthes.

Aschelminthes.
• The animals in which the body cavity is absent are called acoelomates, e.g., Platyhelminthes.
Segmentation
• In some animals, the body is externally and internally divided into segments with a serial
repetition of at least some organs.
• For example, in earthworm, the body shows this pattern called metameric segmentation and
the phenomenon is known as metamerism.
Notochord
• Notochord is a mesodermally [the middle layer of cells or tissues of an embryo, or the parts
derived from this (e.g. cartilage, muscles, and bone)] derived rod-like structure formed on the
dorsal side [posterior] during embryonic development in some animals.
• Animals with notochord are called chordates and those animals which do not form this
structure are called non-chordates, e.g., Porifera to Echinoderms.
Classification of Animal Kingdom
Animal Kingdom is classified into:
1. Phylum – Porifera
2. Phylum – Coelenterata (Cnidaria)
3. Phylum – Ctenophora
4. Phylum – Platyhelminthes
5. Phylum – Aschelminthes (Nemotoda) Annelida
6. Phylum – Arthropoda
7. Phylum – Mollusca
8. Phylum – Echinodermata
9. Phylum – Hemichordata
10. Phylum – Chordata
Phylum – Porifera
• Phylum – Porifera includes organisms with holes.
• They are primitive multicellular animals and have cellular level of organisation.
• They are non-motile animals attached to some solid support.
• The body design involves very minimal differentiation and division into tissues.
• They are commonly called sponges.
• They are generally marine and mostly asymmetrical animals.
• Sponges have a water transport or canal system.
• Water enters through minute pores (ostia) in the body wall into a central cavity, spongocoel,
from where it goes out through the osculum.
• This pathway of water transport is helpful in food gathering, respiratory exchange and removal
of waste.
• The body is supported by a skeleton made up of spicules or spongin fibres.
• Sexes are not separate (hermaphrodite), i.e., eggs and sperms are produced by the same
individual.
• Sponges reproduce asexually by fragmentation and sexually by formation of gametes.
• Fertilisation is internal and development is indirect having a larval stage which is
morphologically distinct from the adult.

Figure: Examples of Porifera : (a) Sycon (b) Euspongia (c) Spongilla
• Examples: Sycon (Scypha), Spongilla (Fresh water sponge) and Euspongia (Bath sponge).
Phylum – Coelenterata (Cnidaria)
• The name cnidaria is derived from the cnidoblasts or cnidocytes (which contain the stinging
capsules or nematocytes) present on the tentacles and the body.
• Cnidoblasts are used for anchorage, defense and for the capture of prey.
• Coelenterata (Cnidaria) are aquatic, mostly marine sessile or free-swimming radially
symmetrical
• They exhibit tissue level of organization [have more body design differentiation than sponges].
• They have a central gastro-vascular cavity with a single opening.
• They are diploblastic.
• Some of these species live in colonies (corals).
• Some have a solitary [living alone] like–span (hydra).
• Some of the cnidarians, e.g., corals have a skeleton composed of calcium carbonate.
• Cnidarians exhibit two basic body forms called polyp and medusa. The former is a sessile and
cylindrical form like Hydra, Adamsia (Sea anemone), etc. whereas, the latter is umbrella-
shaped and free-swimming like Aurelia or jelly fish.
• Those cnidarians which exist in both forms exhibit alternation of generation (Metagenesis),
i.e., polyps produce medusae asexually and medusae form the polyps sexually (e.g., Obelia).
• Jellyfish and sea anemones are common examples.
• Digestion is extracellular and intracellular.
• Examples: Aurelia (jelly fish), Physalia (Portuguese man-of-war), Adamsia (Sea anemone),
Pennatula (Sea-pen), Gorgonia (Sea-fan) and Meandrina (Brain coral).
Phylum – Ctenophora
• Ctenophora are commonly known as sea walnuts or comb jellies.
• They exclusively marine, radially symmetrical, diploblastic
• They exhinit tissue level of organisation.
• The body bears eight external rows of ciliated comb plates, which help in locomotion.
• Digestion is both extracellular and intracellular.
• Bioluminescence (the property of a living organism to emit light) is well-marked in
ctenophores.

ctenophores.
• Sexes are not separate and reproduction takes place only by sexual means.
• Fertilisation is external [fertilization occurs outside the body] with indirect development
[zygote → larvae → animal].
• Examples: Pleurobrachia and Ctenoplana.
Phylum – Platyhelminthes
• Platyhelminthes are more complexly designed than the earlier groups.
• They are bilaterally symmetrical.
• They are triploblastic. This allows outside and inside body linings as well as some organs to be
made. There is thus some degree of tissue formation [organ level of organisation].
• The body is flattened dorsiventrally, meaning from top to bottom, which is why these animals
are called flatworms.
• They may be freeliving or parasitic. Hooks and suckers are present in the parasitic forms.
• Some examples are freeliving animals like planarians, or parasitic animals like
• Parisites are mostly endoparasites found in animals including human beings. Some of them
absorb nutrients from the host directly through their body surface.
• Acoelomate: There is no true internal body cavity or coelom, in which well developed organs
can be accommodated.
• Specialised cells called flame cells help in osmoregulation and excretion.
• Sexes are not separate.
• Fertilisation is internal and development is indirect.
• Some members like Planaria possess high regeneration capacity.

Phylum – Aschelminthes (Nemotoda)
• Body in aschelminthes (Nemotoda) is cylindrical [bilaterally symmetrical] rather than
flattened.
• They exhibit organ-system level of body organization [there are tissues, but no real organs].
• They are triploblastic. A sort of body cavity or a pseudocoelom, is present.
• They are freeliving, aquatic, terrestrial or parasitic in plants and animals.
• These are very familiar as parasitic worms causing diseases, such as the worms causing
elephantiasis (filarial worms) or the worms in the intestines (roundworm or pinworms).
• The body is circular in cross-section, hence, the name roundworms.
• Alimentary canal is complete.
• An excretory tube removes body wastes from the body cavity through the excretory pore.
• Sexes are separate (dioecious), i.e., males and females are distinct.
• Often females are longer than males.
• Fertilisation is internal and development may be direct (the young ones resemble the adult) or
indirect.
Phylum – Annelida
• Annelida are aquatic [marine and fresh water] or terrestrial; free-living, and sometimes
parasitic.
• Their body surface is distinctly marked out into segments or metameres [metamerically

• Their body surface is distinctly marked out into segments or metameres [metamerically
segmented] and, hence, the phylum name Annelida (Latin, annulus: little ring).
• They exhibit organ-system level of body organization.
• They are coelomate [true body cavity]. This allows true organs to be packaged in the body
structure.
• They are bilateral symmetric and triploblastic.
• They possess longitudinal and circular muscles which help in locomotion.
• Aquatic annelids like Nereis possess lateral appendages, parapodia, which help in swimming.
• A closed circulatory system is present.
• Nephridia (sing. nephridium) help in osmoregulation and excretion.
• Neural system consists of paired ganglia (sing. ganglion) connected by lateral nerves to a
double ventral nerve cord.
• Nereis, an aquatic form, is dioecious [Sexes are separate], but earthworms and leeches are
monoecious [having both the male and female reproductive organs in the same individual].
• Reproduction is sexual.
Phylum – Arthropoda
• Insects, arachnids and crustaceans are members of the largest category of creatures on the
planet: arthropods.
• Arthropods have hard, external shells called “exoskeletons,” segmented bodies and jointed
legs.
• Some familiar examples are prawns, butterflies, houseflies, spiders, scorpions and crabs and
some
• They exhibit organ-system level of organisation.
• They are bilaterally symmetrical, triploblastic, segmented and coelomate The coelomic cavity
is blood-filled.
• The body of arthropods is covered by chitinous The body consists of head, thorax and
abdomen.
• There is an open circulatory system, and so the blood does not flow in well defined blood
vessels.
• Respiratory organs are gills, book gills, book lungs or tracheal system.
• Sensory organs like antennae, eyes (compound and simple), statocysts or balance organs are
present.
• Excretion takes place through malpighian tubules.
• They are mostly dioecious.
• Fertilisation is usually internal.
• They are mostly oviparous.
• Development may be direct or indirect.

• Development may be direct or indirect.
Arachnids
• Spiders, harvestmen, mites, ticks and other arachnids are members of the class Arachnida.
Crustaceans
• Crustaceans make up a large group of arthropods that includes animals such as crabs, lobsters,
crayfish and shrimp. They breathe with gills and have two pairs of antennae.
Insects
• In general, insects have three-part bodies, six jointed legs, compound eyes and two antennae.
• Bees, wasps, beetles, mosquitoes, flies, grasshoppers, ants, butterflies and moths, and
dragonflies and damselflies are common types of insects.
Phylum – Mollusca
• Mollusca are the second largest animal phylum. They are terrestrial or aquatic.
• They exhibit organ-system level of organization.
• They are bilaterally symmetrical, triploblastic, coelomate animals. There is little
segmentation.
• They have an open circulatory system and kidney-like organs for excretion. The anterior head
region has sensory tentacles.
• The mouth contains a file-like rasping organ for feeding, called radula.
• They are usually dioecious and oviparous with indirect development.
• Body is covered by a calcareous shell and is unsegmented with a distinct head, muscular foot
and visceral hump. A soft and spongy layer of skin forms a mantle over the visceral hump.
• Examples are octopus, snails and mussels.
Phylum – Echinodermata
• These animals have an endoskeleton of calcareous ossicles [calcium carbonate structures] and,
hence, the name Echinodermata (spiny skinned organisms).
• They are exclusively free-living marine animals with organ-system level of organisation.
• They are triploblastic with a coelomic cavity [coelomate animals]. The adult echinoderms are
radially symmetrical but larvae are bilaterally symmetrical.
• Water-driven tube system [water vascular system] are used for locomotion, capture and

• Water-driven tube system [water vascular system] are used for locomotion, capture and
transport of food and respiration.
• They are triploblastic and coelomate animals.
• Digestive system is complete. An excretory system is absent.
• Sexes are separate. Reproduction is sexual. Fertilisation is usually external.
• Development is indirect with free-swimming larva.
• Examples: Star fish, Sea urchin, Sea lily, Sea cucumber, Brittle star.
Phylum – Hemichordata
• Hemichordata was earlier considered as a sub-phylum under phylum Chordata. But now it is
placed as a separate phylum under non-chordata.
• This phylum consists of a small group of worm-like marine animals with organ-system level of
organisation.
• They are cylindrical [bilaterally symmetrical], triploblastic, coelomate animals.
• The body is Circulatory system is of open type.
• Respiration takes place through gills.
• Excretory organ is present.
• Sexes are separate. Fertilisation is external. Development is indirect.
• Examples: Balanoglossus and Saccoglossus.
Phylum – Chordata
• Animals belonging to phylum Chordata are fundamentally characterised by the presence of a
notochord, a dorsal hollow nerve cord and paired pharyngeal gill slits.
• They are bilaterally symmetrical, triploblastic, coelomate with organ-system level of
organisation.
• They possess a post anal tail and a closed circulatory system.
• Phylum Chordata is divided into three subphyla: Urochordata or Tunicata, Cephalochordata
and Vertebrata.
• Subphyla Urochordata and Cephalochordata are often referred to as protochordates and are
exclusively marine.
• In Urochordata, notochord is present only in larval tail, while in Cephalochordata, it extends
from head to tail region and is persistent throughout their life.
• Examples: Urochordata – Ascidia, Salpa, Doliolum; Cephalochordata – Amphioxus or Lancelet.

All chordates possess the following features:
1. have a notochord
2. have a dorsal nerve cord
3. are triploblastic
4. have paired gill pouches
5. are coelomate.
Vertebrata
• These animals have a true vertebral column and internal skeleton, allowing a completely
different distribution of muscle attachment points to be used for movement.
• The members of subphylum Vertebrata possess notochord during the embryonic period.
• The notochord is replaced by a cartilaginous or bony vertebral column in the adult.
• Thus all vertebrates are chordates but all chordates are not vertebrates.
• Besides the basic chordate characters, vertebrates have a ventral muscular heart with two,
three or four chambers, kidneys for excretion and osmoregulation and paired appendages
which may be fins or limbs.
• Vertibrates are bilaterally symmetrical, triploblastic, coelomic and segmented, with complex
differentiation of body tissues and organs.
Comparison of Chordates
and Non-chordates
S.No. Chordates Non-chordates
1. Notochord present. Notochord absent.
2. Central nervous system is Central nervous system is
dorsal, hollow and single. ventral, solid and double.
3. Pharynx perforated by gill slits. Gill slits are absent.
4. Heart is ventral. Heart is dorsal (if present).
5. A post-anal part (tail) is Post-anal tail is absent.
present.
From <https://www.pmfias.com/classification-animalia-animal-kingdom/>

Vertebrate Classification
Vertebrata
• These animals have a true vertebral column and internal skeleton, allowing a completely
different distribution of muscle attachment points to be used for movement.
• The members of subphylum Vertebrata possess notochord during the embryonic period.
• The notochord is replaced by a cartilaginous or bony vertebral column in the adult.
• Thus all vertebrates are chordates but all chordates are not vertebrates.
• Besides the basic chordate characters, vertebrates have a ventral muscular heart with two,
three or four chambers, kidneys for excretion and osmoregulation and paired appendages
which may be fins or limbs.
• Vertibrates are bilaterally symmetrical, triploblastic, coelomic and segmented, with complex

Division In Vertebrata

Basic Concepts
Viviparous and Oviparous Animals
• We have learnt that some animals give birth to young ones while some animals lay eggs which
later develop into young ones.
• The animals which give birth to young ones are called viviparous animals.
• Those animals which lay eggs are called oviparous animals.
• In some animals, the young ones may look very different from the adults. Recall the life cycle
of the silkworm (egg → larva or caterpillar → pupa → adult) (egg → tadpole (larva) → adult).
The transformation of the larva into an adult through drastic changes is called
metamorphosis.
Warm Blooded vs. Cold Blooded Animals
Warm Blooded or Endotherms or Homoiothermous Cold Blooded or Ectotherms or
animals Poikilothermous Animals
• All mammals and birds with few exceptions • All reptiles, insects, arachnids,
are warm blooded. [Bats, Echidnas, Mole Rats amphibians and fish are cold
etc. cannot regulate their body temperature] blooded.
• They maintain a constant internal body • Their body temperature changes
temperature irrespective of external according to the external
environment. [Can regulate their body environment. [If a cold blooded
temperature by generating their own heat animal is taken to the equator its
when they are in a cooler environment, and body temperature increases and if
by cooling themselves when they are in a taken to the poles its body
hotter environment] temperature decreases]
• They can survive in a wide of environments as • They cannot survive in a wide of
they are able to regulate their body environments. [Tropical animals
temperature. cannot survive in the polar region
and vice versa]
• They require a lot of food for their survival. • Most of the food consumed is
Most of the food consumed is utilized to converted into body mass. So they
maintain a constant body temperature. need less food compared to warm
blooded animals.
• They are active in both warm and cold • They are active in warm
environments. environments and are very sluggish in
cold environments.
• To stay cool, warm-blooded animals usually • Cold-blooded animals often like to
sweat. Animals like elephants use their ears bask in the sun to warm up and
to cool their body [large, thin ears which increase their metabolism.
loose heat quickly]. • Some cold-blooded animals, such as
• Some warm-blooded animals, especially bees or dragonflies, shiver to stay
birds, migrate from colder to warmer regions warm when in a cold environment.
in the winter.
• Mammals have hair, fur and birds have
feathers to help keep them warm.
• Warm-blooded animals can also shiver to
generate more heat when they get too cold.
• Constant body temperature provide a nice • Constantly changing body
warm environment for viruses, bacteria and temperatures make life more difficult
parasites to live in. for the parasites.
Hibernation
• Hibernation is a state of inactivity and metabolic depression in few endotherms [warm
blooded animals – bear, rodents] and ectotherms [many reptiles like snakes, turtles and
amphibians like frogs]. Snakes, lizards, toads, frogs, salamanders and most turtles will mostly
hibernate during harsh winters.
• Hibernating animals usually retreat to a den, a burrow, or a hollow log for protection and
shelter.
• During “true hibernation,” the animal’s body temperature drops, and its rate of breathing
slows down. These hibernating animals are very difficult to awaken.
• Some warm-blooded animals such as bears, rodents etc. hibernate during extreme weather
seasons and unfavorable conditions.
• During hibernation these animals live off of stored body fat and can drop their body
temperatures significantly.
• Most animals will eat large amounts of food before hibernating.
Class – Cyclostomata
Class – Cyclostomata
• All living members of the class Cyclostomata are ectoparasites [ives on the outside of its host]
on some fishes.
• They have an elongated body bearing 6-15 pairs of gill slits for respiration.
• Cyclostomes have a sucking and circular mouth without jaws.
• Their body is devoid of scales and paired fins.
• Cranium and vertebral column are cartilaginous.
• Circulation is of closed type.
• Cyclostomes are marine but migrate for spawning [release or deposit eggs] to fresh water.
• After spawning, within a few days, they die. Their larvae, after metamorphosis
[transformation from an immature form to an adult form in two or more distinct stages.
Example: Larvae → Tadpole → Frog], return to the ocean.
• Examples: Petromyzon (Lamprey) and Myxine (Hagfish).
Class – Pisces
• These are fish. Their skin is covered with scales/plates. They lay eggs [oviporous].
• They obtain oxygen dissolved in water by using gills.
• The body is streamlined, and a muscular tail is used for movement.
• They are cold-blooded and their hearts have only two chambers, unlike the four that humans
have.
• Some fish skeletons are made entirely of cartilage [Chondrichthyes], such as sharks, and some
with a skeleton made of both bone and cartilage [Osteichthyes].
Chondrichthyes
• They are marine animals with streamlined body and have cartilaginous endoskeleton. Mouth
is located ventrally.
• Notochord is persistent throughout life.
• Gill slits are separate and without operculum (gill cover).
• The skin is tough, containing minute placoid scales.
• Teeth are modified placoid scales which are backwardly directed.
• Their jaws are very powerful.
• These animals are predaceous [shark].
• Due to the absence of air bladder, they have to swim constantly to avoid sinking.
• Heart is two-chambered (one auricle and one ventricle).
• Some of them have electric organs (e.g., Torpedo) and some possess poison sting (e.g.,
Trygon).
• They are cold-blooded (poikilothermous) animals, i.e., they lack the capacity to regulate their
body temperature.
• Sexes are separate. In males pelvic fins bear claspers.
• They have internal fertilisation and many of them are viviparous [give birth to young ones].
• Examples: Scoliodon (Dog fish), Pristis (Saw fish), Carchaiodon (Great white shark), Trygon
(Sting ray).
Osteichthyes
• It includes both marine and fresh water fishes with bony endoskeleton.
• Their body is streamlined. Mouth is mostly terminal.
• They have four pairs of gills which are covered by an operculum on each side.
• Skin is covered with cycloid/ctenoid scales.
• Air bladder is present which regulates buoyancy.
• Heart is two- chambered (one auricle and one ventricle).
• They are cold-blooded
• Sexes are separate.
• Fertilisation is usually external.
• They are mostly oviparous and development is direct.
• Examples: Flying fish, Sea horse, Fighting fish, Angel fish etc.

Class – Amphibia
• As the name indicates (Gr., Amphi : dual, bios, life), amphibians can live in aquatic as well as
terrestrial habitats.
• The amphibian skin is moist without scales [mucus glands in the skin]. The eyes have eyelids. A
tympanum represents the ear.
• Alimentary canal, urinary and reproductive tracts open into a common chamber called cloaca
which opens to the exterior.
• They have a three-chambered heart (two auricles and one ventricle). These are cold-blooded
• Respiration is through gills, lungs and through
• Respiration is by gills, lungs and through skin.
• Sexes are separate. Fertilisation is external.
• They are oviparous and development is indirect.
• Examples: Toad, Frog), Tree frog, Salamander, Limbless amphibia.
Class – Reptilia
• The class name refers to their creeping or crawling mode of locomotion (Latin, repere or
reptum, to creep or crawl).
• They are mostly terrestrial animals and their body is covered by dry and cornified skin,
epidermal scales or scutes. Snakes and lizards shed their scales as skin cast.
• They do not have external ear openings. Tympanum represents ear. Limbs, when present, are
two pairs.
• Heart is usually three-chambered, but four-chambered in crocodiles.
• Reptiles are poikilotherms [cold-blooded animals].
• They lay eggs with tough coverings and do not need to lay their eggs in water, unlike
amphibians.
• Sexes are separate.
• Fertilisation is internal.
• They are oviparous and development is direct.
• Examples: Turtle), Tortoise, Chameleon (Tree lizard), Garden lizard, Crocodile, Alligator, Wall
lizard, Poisonous snakes – Naja (Cobra), Bangarus (Krait), Vipera (Viper).
Class – Aves
• They have a four-chambered heart. They breathe through lungs. All birds fall in this category.
• The characteristic features of Aves (birds) are the presence of feathers and most of them can
fly except flightless birds (e.g., Ostrich). The forelimbs are modified into wings.
• The hind limbs generally have scales and are modified for walking, swimming or clasping the

• The hind limbs generally have scales and are modified for walking, swimming or clasping the
tree branches.
• Skin is dry without glands except the oil gland at the base of the tail.
• Endoskeleton is fully ossified (bony) and the long bones are hollow with air cavities
(pneumatic).
• The digestive tract of birds has additional chambers, the crop and gizzard.
• They are warm-blooded (homoiothermous) animals, i.e., they are able to maintain a constant
body temperature.
• Respiration is by lungs. Air sacs connected to lungs supplement respiration.
• Sexes are separate. Fertilisation is internal. They are oviparous and development is direct.
• Examples : Crow, Pigeon, Ostrich), Neophron (Vulture) etc..
Class – Mammalia
• Mammals are warm-blooded animals with four-chambered hearts.
• Most mammals familiar to us produce live young ones. However, a few of them, like the
Platypus and the Echidna lay eggs, and some, like kangaroos give birth to very poorly
developed young ones.
• They are found in a variety of habitats – polar ice caps, deserts, mountains, forests, grasslands
and dark caves. Some of them have adapted to fly or live in water.
• The most unique mammalian characteristic is the presence of milk producing glands
(mammary glands) by which the young ones are nourished.
• They have two pairs of limbs, adapted for walking, running, climbing, burrowing, swimming or
flying.
• The skin of mammals is unique in possessing hair. External ears or pinnae are present.
Different types of teeth are present in the jaw.
• Heart is four-chambered. They are homoiothermous [warm-blooded]. Respiration is by lungs.
• Sexes are separate and fertilisation is internal.
• They are viviparous with few exceptions and development is direct.
• Examples: Oviparous – Platypus; Viviparous – Kangaroo, Flying fox), Delphinus (Common
dolphin), Balaenoptera (Blue whale), etc.
Animal Classification Summary
• Porifera includes multicellular animals which exhibit cellular level of organisation and have
characteristic flagellated choanocytes.
• The coelenterates have tentacles and bear cnidoblasts. They are mostly aquatic, sessile or
free-floating. The ctenophores are marine animals with comb plates.
• The platyhelminths have flat body and exhibit bilateral symmetry. The parasitic forms show
distinct suckers and hooks.
• Aschelminthes are pseudocoelomates and include parasitic as well as non-parasitic round
worms.
• Annelids are metamerically segmented animals with a true coelom.
• The arthropods are the most abundant group of animals characterised by the presence of
jointed appendages.
• The molluscs have a soft body surrounded by an external calcareous shell. The body is covered
with external skeleton made of chitin.
• The echinoderms possess a spiny skin. Their most distinctive feature is the presence of water
vascular system.
• The hemichordates are a small group of worm-like marine animals. They have a cylindrical
body with proboscis, collar and trunk.
• Phylum Chordata includes animals which possess a notochord either throughout or during
early embryonic life. Other common features observed in the chordates are the dorsal, hollow
nerve cord and paired pharyngeal gill slits.
• Some of the vertebrates do not possess jaws (Agnatha) whereas most of them possess jaws
(Gnathostomata). Agnatha is represented by the class, Cyclostomata. They are the most
primitive chordates and are ectoparasites on fishes. Gnathostomata has two super classes,
Pisces and Tetrapoda.
• Classes Chondrichthyes and Osteichthyes bear fins for locomotion and are grouped under
Pisces. The Chondrichthyes are fishes with cartilaginous endoskeleton and are marine.
• Classes, Amphibia, Reptilia, Aves and Mammalia have two pairs of limbs and are thus grouped
under Tetrapoda. The amphibians have adapted to live both on land and water.
• Reptiles are characterised by the presence of dry and cornified skin. Limbs are absent in
snakes. Fishes, amphibians and reptiles are poikilothermous (coldblooded).
• Aves are warm-blooded animals with feathers on their bodies and forelimbs modified into
wings for flying. Hind limbs are adapted for walking, swimming, perching or clasping.
• The unique features of mammals are the presence of mammary glands and hairs on the skin.
They commonly exhibit viviparity.
Match the following
1. Operculum a) Ctenophora
2. Parapodia b) Mollusca
3. Scales c) Porifera
4. Comb plates d) Reptilia
5. Radula e) Annelida
6. Hairs f) Cyclostomata and Chondrichthyes
7. Choanocytes g) Mammalia
8. Gill slits h) Osteichthyes
Salient
Features
of
Different
Phyla in
the
Animal
Kingdom
Phylum Level of Symmet Coelom Segmen Digestiv Circu- Respi- Distinctive
Organi- ry tion e latory ratory Features

Organi- ry tion e latory ratory Features
sation System System System
Porifera Cellular Various Absent Absent Absent Absent Absent Body with
pores and
canals in
walls.
Coelentera Tissue Radial Absent Absent Incompl Absent Absent Cnidoblasts
ta ete present.
(Cnidaria)
Ctenophor Tissue Radial Absent Absent Incompl Absent Absent Comb
a ete plates for
locomotion
.
Platyhelm- Organ & Bilatera Absent Absent Incompl Absent Absent Flat body,
inthes Organ – l ete suckers.
system
Aschelmin- Organ – Bilatera Pseudo- Absent Comple Absent Absent Often
thes system l coeloma te worm-
te shaped,
elongated.
Annelida Organ – Bilatera Coelom Present Comple Present Absent Body
system l ate te segment-
ation like
rings.
Arthropoda Organ – Bilatera Coelom Present Comple Present Present Exoskeleto
system l ate te n of cuticle,
jointed ap-
pendages.
Mollusca Organ – Bilatera Coelom Absent Comple Present Present External
system l ate te skeleton of
shell
usually
present.
Echino- Organ- Radial Coelom Absent Comple Present Present Water
dermata system ate te vascular
system,
radial
symmetry.
Hemi- Organ- Bilatera Coelom Absent Comple Present Present Worm-like
chordata system l ate te with
proboscis,
collar and
trunk.
Chordata Organ- Bilatera Coelom Present Comple Present Present Notochord,
system l ate te dorsal
hollow
nerve cord,
gill slits
with limbs
or fins.
From <https://www.pmfias.com/classification-vertebrata-phylum-chordata/>

Plant Kingdom – Thallophytes (Algae) – Bryophytes –
Pteridophytes
PLANTAE
• Classification among plants depends on
1. whether the plant body has well differentiated, distinct components,
2. whether the differentiated plant body has special tissues for the transport of water and other
substances within it,
3. ability to bear seeds, and
4. whether the seeds are enclosed within fruits.
• Phylogenetic classification [evolutionary relationships], cytotaxonomy [cytological
information like chromosome number, structure, behavior] and chemotaxonomy [chemical
constituents of the plant], are used by taxonomists for classifying plants.
Plant Kingdom
• Plants are multicellular eukaryotes with cell walls mainly made of cellulose {Plant Cell vs.
Animal Cell}.
• They are autotrophs and use chlorophyll for photosynthesis. A few members are partially

• They are autotrophs and use chlorophyll for photosynthesis. A few members are partially
heterotrophic such as the insectivorous plants or parasites. Bladderwort and Venus fly trap
are examples of insectivorous plants and Cuscuta is a parasite.
• Plantae includes algae, bryophytes, pteridophytes, gymnosperms and angiosperms.
• Fungi, and members of the Monera and Protista having cell walls have now been excluded
from Plantae. So, the cyanobacteria that are also referred to as blue green algae are not
‘algae’ any more.
Algae – Thallophytes
• Plants that do not have well-differentiated body design fall in this group. They are commonly
called algae.
• Algae are chlorophyll-bearing, simple, thalloid, autotrophic and largely aquatic (both fresh
water and marine) organisms.
[Thallus == a plant body not differentiated into stem, leaves, and roots and without a vascular
system, typical of algae, fungi, lichens, and some liverworts].
• They occur in a variety of other habitats: moist stones, soils and wood. Some of them also
occur in association with fungi (lichen) and animals (e.g., on sloth bear).
• The form and size of algae is highly variable. The size ranges from the microscopic unicellular
forms like Chlamydomonas, to colonial forms like Volvox and to the filamentous forms like
Ulothrix and Spirogyra. A few of the marine forms such as kelps, form massive plant bodies.
• The algae reproduce by vegetative, asexual and sexual methods. Vegetative reproduction is
by fragmentation. Each fragment develops into a thallus.
• Asexual reproduction is by the production of different types of spores, the most common
being the zoospores [capable of swimming by means of a flagellum]. They are flagellated
(motile) and on germination gives rise to new plants.
• Sexual reproduction takes place through fusion of two gametes. These gametes can be
flagellated and similar in size (as in Chlamydomonas) or non-flagellated (non-motile) but
similar in size (as in Spirogyra). Such reproduction is called isogamous [Fusion of two gametes
similar in size].
• Fusion of two gametes dissimilar in size, as in some species of Chlamydomonas is termed as
anisogamous.
• Fusion between one large, non-motile (static) female gamete and a smaller, motile male
gamete is termed oogamous, e.g., Volvox, Fucus. [Compare this with human sperm and ovum]
Chlorophyceae – Green Algae

• The members of chlorophyceae are commonly called green algae.
• The plant body may be unicellular, colonial or filamentous.
• They are usually grass green due to the dominance of pigments chlorophyll a and b. The
pigments are localised in definite chloroplasts.
• Most of the members have one or more storage bodies called pyrenoids located in the
chloroplasts. Pyrenoids contain protein besides starch. Some algae may store food in the form
of oil droplets.
• Green algae usually have a rigid cell wall made of an inner layer of cellulose and an outer layer
of pectose.
• Vegetative reproduction usually takes place by fragmentation or by formation of different
types of spores.
• Asexual reproduction is by flagellated zoospores produced in zoosporangia.
• The sexual reproduction shows considerable variation in the type and formation of sex cells
and it may be isogamous, anisogamous or oogamous.
• Some commonly found green algae are: Chlamydomonas, Volvox, Ulothrix, Spirogyra and
Chara.
Phaeophyceae – Brown Algae

• The members of phaeophyceae or brown algae are found primarily in marine habitats.
• They show great variation in size and form. They range from simple branched, filamentous
forms (Ectocarpus) to profusely branched forms as represented by kelps, which may reach a
height of 100 metres.
• They possess chlorophyll a, c, carotenoids and xanthophylls. They vary in colour from olive
green to various shades of brown depending upon the amount of the xanthophyll pigment,
fucoxanthin present in them.
• The vegetative cells have a cellulosic wall usually covered on the outside by a gelatinous
coating of algin. The protoplast contains, in addition to plastids, a centrally located vacuole
and nucleus.
• Vegetative reproduction takes place by fragmentation.

• Vegetative reproduction takes place by fragmentation.
• Asexual reproduction in most brown algae is by biflagellate zoospores that are pear-shaped
and have two unequal laterally attached flagella.
• Sexual reproduction maybe isogamous, anisogamous or oogamous.
• Union of gametes may take place in water or within the oogonium (oogamous species).
• The gametes are pyriform (pear-shaped) and bear two laterally attached flagella.
• The common forms are Ectocarpus, Dictyota, Laminaria, Sargassum and Fucus.
Rhodophyceae – Red Algae

• The members of rhodophyceae are commonly called red algae because of the predominance
of the red pigment, r-phycoerythrin in their body.
• Majority of the red algae are marine with greater concentrations found in the warmer areas.
• They occur in both well-lighted regions close to the surface of water and also at great depths
in oceans where relatively little light penetrates.
• The red thalli of most of the red algae are multicellular. Some of them have complex body
organisation.
• The food is stored as floridean starch which is very similar to amylopectin and glycogen in
structure.
• The red algae usually reproduce vegetatively by fragmentation.
• They reproduce asexually by non-motile spores and sexually by non-motile gametes.
• Sexual reproduction is oogamous.
• The common members are: Polysiphonia, Porphyra, Gracilaria and Gelidium.
Uses of algae
• Algae are useful to man in a variety of ways. At least a half of the total carbon dioxide fixation
on earth is carried out by algae through photosynthesis.
• Being photosynthetic they increase the level of dissolved oxygen in their immediate
environment.
• They are of paramount importance as primary producers of energy-rich compounds which
form the basis of the food cycles of all aquatic animals.
• Many species of Porphyra, Laminaria and Sargassum are among the 70 species of marine
algae used as food.
• Certain marine brown and red algae produce large amounts of hydrocolloids (water holding
substances), e.g., algin (brown algae) and carrageen (red algae) which are used commercially.
• Agar, one of the commercial products obtained from Gelidium and Gracilaria are used to grow
microbes and in preparations of ice-creams and jellies.
• Chlorella a unicellular alga, rich in proteins is used as food supplement even by space
travellers.
• The algae are divided into three main classes: Chlorophyceae, Phaeophyceae and
Rhodophyceae.
Bryophytes
• Bryophytes are called amphibians of the plant kingdom because these plants can live in soil
but are dependent on water for sexual reproduction.
• The plant body is commonly differentiated to form stem and leaf-like structures. However,
there is no specialized tissue for the conduction of water and other substances from one part
of the plant body to another.
• Bryophytes include the various mosses (funaria), marchantia and liverworts that are found
commonly growing in damp, humid and shaded localities. They play an important role in plant
succession on bare rocks/soil.

• The plant body of bryophytes is more differentiated than that of algae. It is thallus-like and
erect, and attached to the substratum by unicellular or multicellular rhizoids [root like
structures].
• They lack true roots, stem or leaves. They may possess root-like, leaf-like or stem-like
structures.
• The main plant body of the bryophyte is haploid. It produces gametes, hence is called a
gametophyte.
• The sex organs in bryophytes are multicellular. The male sex organ is called antheridium. They
produce biflagellate antherozoids. The female sex organ called archegonium is flask-shaped
and produces a single egg.
• The antherozoids are released into water where they come in contact with archegonium. An
antherozoid fuses with the egg to produce the zygote.
• Zygotes do not undergo reduction division [Meiosis] immediately. They produce a multicellular
body called a sporophyte. The sporophyte is not free-living but attached to the photosynthetic
gametophyte and derives nourishment from it.
• Some cells of the sporophyte undergo reduction division (meiosis) to produce haploid spores.
These spores germinate to produce gametophyte.
• Bryophytes in general are of little economic importance but some mosses provide food for
herbaceous mammals, birds and other animals.
• Species of Sphagnum, a moss, provide peat that have long been used as fuel, and as packing
material for trans-shipment of living material because of their capacity to hold water.
• Mosses along with lichens are the first organisms to colonise rocks and hence, are of great
ecological importance. They decompose rocks making the substrate suitable for the growth of
higher plants.
• Since mosses form dense mats on the soil, they reduce the impact of falling rain and prevent
soil erosion. The bryophytes are divided into liverworts and mosses.
Pteridophytes
• In this group, the plant body is differentiated into roots, stem and leaves and has specialized
tissue for the conduction of water and other substances from one part of the plant body to
another. Some examples are marsilea, ferns and horse-tails.
• Pteridophytes are used for medicinal purposes and as soil-binders. They are also frequently
grown as ornamentals.
• Evolutionarily, they are the first terrestrial plants to possess vascular tissues – xylem and
phloem.
• The pteridophytes are found in cool, damp, shady places though some may flourish well in
sandy-soil conditions.
• You may recall that in bryophytes the dominant phase in the life cycle is the gametophytic
plant body. However, in pteridophytes, the main plant body is a sporophyte which is
differentiated into true root, stem and leaves.
• These organs possess well-differentiated vascular tissues. The leaves in pteridophyta are small
(microphylls) as in Selaginella or large (macrophylls) as in ferns.
• The spores germinate to give rise to inconspicuous, small but multicellular, free-living, mostly
photosynthetic thalloid gametophytes called prothallus.
• These gametophytes require cool, damp, shady places to grow. Because of this specific
restricted requirement and the need for water for fertilisation, the spread of living
pteridophytes is limited and restricted to narrow geographical regions.
Cryptogamae
• The thallophytes, the bryophytes and the pteridophytes have naked embryos that are called
spores.
• The reproductive organs of plants in all these three groups are very inconspicuous, and they
are therefore called ‘cryptogamae’, or ‘those with hidden reproductive organs’.
From <https://www.pmfias.com/plant-kingdom-thallophytes-algae-bryophytes-pteridophytes/>

From <https://www.pmfias.com/plant-kingdom-thallophytes-algae-bryophytes-pteridophytes/>

Plants with Seeds – Gymnosperms and Angiosperms
Phanerogams – Plants with Seeds

• Plants with well differentiated reproductive tissues that ultimately make seeds are called
phanerogams.
• Seeds are the result of the reproductive process. They consist of the embryo along with stored
food, which serves for the initial growth of the embryo during germination.
• This group is further classified, based on whether the seeds are naked or enclosed in fruits,
giving us two groups: gymnosperms and angiosperms.
Gymnosperms
• This term is made from two greek words: gymno– means naked and sperma– means seed.
• The plants of this group bear naked seeds [ovules are not enclosed by any ovary wall] and are
usually perennial, evergreen and woody. The seeds that develop post-fertilisation are naked
too. Examples are pines, such as deodar.

too. Examples are pines, such as deodar.
• Gymnosperms include medium-sized trees or tall trees and shrubs. One of the gymnosperms,
the giant redwood tree Seguoia is one of the tallest tree species.
• The roots are generally tap roots {Plant Parts and Their Functions}. Roots in some genera have
fungal association in the form of mycorrhiza (Pinus), while in some others (Cgcas) small
specialised roots called coralloid roots are associated with N2-fixing cyanobacteria.
• The leaves in gymnosperms are well-adapted to withstand extremes of temperature, humidity
and wind.
• In conifers, the needle-like leaves reduce the surface area. Their thick cuticle and sunken
stomata also help to reduce water loss.
• The gymnosperms are heterosporous; they produce haploid microspores and megaspores.
• The two kinds of spores are produced within sporangia that are borne on sporophylls which
are arranged spirally along an axis to form lax or compact strobili or cones.
• The strobili bearing microsporophylls and microsporangia are called microsporangiate or male
strobili.
• The microspores develop into a male gametophytic generation which is highly reduced and is
confined to only a limited number of cells. This reduced gametophyte is called a pollen grain.
The development of pollen grains take place within the microsporangia.
• The cones bearing megasporophylls with ovules or megasporangia are called
macrosporangiate or female strobili.
• The male or female cones or strobili may be borne on the same tree (Pinus). However, in cycas
male cones and megasporophylls are borne on different trees.
• Unlike bryophytes and pteridophytes {Bryophytes – Pteridophytes }, in gymnosperms the male
and the female gametophytes do not have an independent free-living existence. They remain
within the sporangia retained on the sporophytes.
• The pollen grain is released from the microsporangium. They are carried in air currents and
come in contact with the opening of the ovules borne on megasporophylls.
• The pollen tube carrying the male gametes grows towards archegonia in the ovules and
discharge their contents near the mouth of the archegonia.
• Following fertilisation, zygote develops into an embryo and the ovules into seeds. These seeds
are not covered.
Figure: Gymnosperms: (a) Cycas (b) Pinus (c) Ginkgo
Angiosperms
• This word is made from two greek words: angio– means covered and sperma– means seed.
• Unlike the gymnosperms where the ovules are naked, in the angiosperms or flowering plants,

• Unlike the gymnosperms where the ovules are naked, in the angiosperms or flowering plants,
the pollen grains and ovules are developed in specialised structures called flowers.
• The seeds develop inside an organ which is modified to become a fruit. These are also called
flowering plants.
• The male sex organ in a flower is the stamen. Each stamen consists of a slender filament with
an anther at the tip. The anthers, following Meiosis, produce pollen grains.
• The female sex organ in a flower is the pistil or the carpel. Pistil consists of an ovary enclosing
one to many ovules. Within ovules are present highly reduced female gametophytes termed
embryo-sacs. The embryo-sac formation is preceded by meiosis. Hence, each of the cells of an
embryo-sac is haploid.
• Each embryo-sac has a three-celled egg apparatus – one egg cell and two synergids, three
antipodal cells and two polar nuclei. The polar nuclei eventually fuse to produce a diploid
secondary nucleus.
• Pollen grain, after dispersal from the anthers, are carried by wind or various other agencies to
the stigma of a pistil. This is termed as pollination.
• The pollen grains germinate on the stigma and the resulting pollen tubes grow through the
tissues of stigma and style and reach the ovule.
• The pollen tubes enter the embryo-sac where two male gametes are discharged. One of the
male gametes fuses with the egg cell to form a zygote (syngamy).
• The other male gamete fuses with the diploid secondary nucleus to produce the triploid
primary endosperm nucleus (PEN).
• Because of the involvement of two fusions, this event is termed as double fertilisation, an
event unique to angiosperms.
Figure: Life cycle of an angiosperm

• The zygote develops into an embryo (with one or two cotyledons) and the PEN develops into
endosperm which provides nourishment to the developing embryo.
• The synergids and antipodals degenerate after fertilisation. During these events the ovules
develop into seeds and the ovaries develop into fruit.
• Plant embryos in seeds have structures called cotyledons. Cotyledons are called ‘seed leaves’
because in many instances they emerge and become green when the seed germinates. Thus,
cotyledons represent a bit of pre-designed plant in the seed.
Monocots and Dicots

• The angiosperms are divided into two groups on the basis of the number of cotyledons
present in the seed.
• Plants with seeds having a single cotyledon are called monocotyledonous or monocots. Plants
with seeds having two cotyledons are called dicots.

Figure: Angiosperms : (a) A dicotyledon (b) A monocotyledon
Kingdom Plantae – Summary

• Plant kingdom includes algae, bryophytes, pteridophytes, gymnosperms and angiosperms.
• Algae [thallophytes] are chlorophyll-bearing simple, thalloid, autotrophic and largely aquatic
organisms.
• Depending on the type of pigment possesed and the type of stored food, algae are classfied
into three classes, namely Chlorophyceae, Phaeophyceae and Rhodophyceae.
• Algae usually reproduce vegetatively by fragmentation, asexually by formation of different
types of spores and sexually by formation of gametes which may show isogamy, anisogamy or
oogamy.
• Bryophytes are plants which can live in soil but are dependent on water for sexual
reproduction. Their plant body is more differentiated than that of algae. It is thallus-like and
prostrate or erect and attached to the substratum by rhizoids. They possess root-like, leaf-like
and stem-like structures.
• The bryophytes are divided into liverworts and mosses. The plant body of liverworts is thalloid
and dorsiventral whereas mosses have upright, slender axes bearing spirally arranged leaves.
• The main plant body of a bryophyte is gamete-producing and is called a gametophyte. It bears
the male sex organs called antheridia and female sex organs called archegonia.
• The male and female gametes produced fuse to form zygote which produces a multicellular
body called a sporophyte. It produces haploid spores. The spores germinate to form
gametophytes.
• In pteridophytes the main plant is a sporophyte which is differentiated into true root, stem
and leaves. These organs possess well-differentiated vascular tissues. The sporophytes bear
sporangia which produce spores.
• The spores germinate to form gametophytes which require cool, damp places to grow. The
gametophytes bear male and female sex organs called antheridia and archegonia, respectively.
Water is required for transfer of male gametes to archegonium where zygote is formed after
fertilisation. The zygote produces a sporophyte.
• The gymnosperms are the plants in which ovules are not enclosed by any ovary wall. After
fertilisation the seeds remain exposed and therefore these plants are called naked-seeded
plants.
• The gymnosperms produce microspores and megaspores which are produced in
microsporangia and megasporangia borne on the sporophylls. The sporophylls –
microsporophylls and megasporophylls – are arranged spirally on axis to form male and female
cones, respectively. The pollen grain germinates and pollen tube releases the male gamete
into the ovule, where it fuses with the egg cell in archegonia. Following fertilisation, the zygote
develops into embryo and the ovules into seeds.
• In angiosperms, the male sex organs (stamen) and female sex organs (pistil) are borne in a
flower. Each stamen consists of a filament and an anther. The anther produces pollen grains
(male gametophyte) after meiosis. The pistil consists of an ovary enclosing one to many
ovules. Within the ovule is the female gametophyte or embryo sac which contains the egg cell.
• The pollen tube enters the embryo-sac where two male gametes are discharged. One male
gamete fuses with egg cell (syngamy) and other fuses with diploid secondary nucleus (triple
fusion). This phenomenon of two fusions is called double fertilisation and is unique to
angiosperms. The angiosperms are divided into two classes – the dicotyledons and the
monocotyledons.
• During the life cycle of any sexually reproducing plant, there is alternation of generations
between gamete producing haploid gametophyte and spore producing diploid sporophyte.
However, different plant groups as well as individuals may show different patterns of life
cycles – haplontic, diplontic or intermediate.
Match the following (column I with column II)
Column I Column II
Chlamydomonas Moss
Cycas Pteridophyte
Selaginella Algae

Selaginella Algae
Sphagnum Gymnosperm
From <https://www.pmfias.com/plants-seeds-gymnosperms-angiosperms/>

Principles of taxonomy
Thursday, 28 October, 2021 08:20 PM

Classification

Common diseases in human beings
Disease can be defined as a disorder or malfunction of the mind or body.
Common diseases in human beings

Disease can be defined as a disorder or malfunction of the mind or body. It involves
morphological, physiological and psychological disturbances which may be due to
environmental factors or pathogens or genetic anomalies or life style changes. Diseases can
be broadly grouped into infectious and non infectious types.
Diseases which are transmitted from one person to another are called infectious diseases or
communicable diseases. Such disease causing organisms are called pathogens and are
transmitted through air, water, food, physical contact and vectors. The disease causing
pathogen may be virus, bacteria, fungi, protozoan parasites, helminthic parasites, etc.,
Infectious diseases are common and everyone suffers from such diseases at some time or
the other. Most of the bacterial diseases are curable but all viral diseases are not. Some
infectious disease like AIDS may be fatal.
Non-infectious diseases are not transmitted from an infected person to a healthy person. In
origin they may be genetic (cystic fibrosis), nutritional (vitamin deficiency diseases) and
degenerative (arthritis, heart attack, stroke). Among non - infectious diseases, cancer is one
of the major causes of death.
To Memorize Page 102

1. Bacterial and viral diseases
Bacterial diseases
Though the number of bacterial species is very high, only a few bacteria are associated with
human diseases and are called pathogenic bacteria. Such pathogens may emit toxins and
affected the body. Common pathogenic bacteria and the bacterial diseases are given in
table 7.1.

Bacteria spread through air, water or by inhaling the droplets/aerosols or even by sharing
utensils, dresses with an infected person. Typhoid fever can be confirmed by Widal test.
Viral diseases
Viruses are the smallest intracellular obligate parasites, which multiply within living cells.
Outside the living cells they cannot carry out the characteristics of a living organism.
Viruses invade living cells, forcing the cells to create new viruses. The new viruses break
out of the cell, killing it and invade other cells in the body, causing diseases in human
beings. Rhino viruses cause one of the most infectious human ailment called the “Common
cold”.

cold”.
SS-RNA
SS-RNA
SS-RNA
DNA -- replicates using reverse transcription
SS-RNA +ve stranded - with protein capsid
SS-RNA +ve stranded
SS-RNA

Viral diseases are generally grouped into four types on the basis of the symptoms produced
in the body organs.
i. Pneumotropic diseases (respiratory tract infected by influenza)
ii. Dermotropic diseases (skin and subcutaneous tissues affected by chicken pox and
measles)
iii. Viscerotropic diseases (blood and visceral organs affected by yellow fever and
dengue fever)
iv. Neurotropic diseases (central nervous system affected by rabies and polio). Some
common viral diseases of human beings are given in table 7.2.
2. Protozoan diseases
About 15 genera of protozoans live as parasites within the human body and cause diseases.
Amoebiasis also called amoebic dysentery or amoebic colitis is caused by Entamoeba
histolytica, which lives in the human large intestine and feeds on food particles and bacteria
(Fig. 7.1). Infective stage of this parasite is the trophozoite, which penetrates the walls of
the host intestine (colon) and secretes histolytic enzymes causing ulceration, bleeding,
abdominal pain and stools with excess mucus. Symptoms of amoebiasis can range from
diarrhoea to dysentery with blood and mucus in the stool. House flies (Musca domestica)
acts as a carrier for transmitting the parasite from contaminated faeces and water.
African sleeping sickness is caused by Trypanosoma species. Trypanosoma is generally

transmitted by the blood sucking Tsetse flies. Three species of Trypanosoma cause sleeping
sickness in man.

1. T. gambiense is transmitted by Glossina palpalis (Tsetse fly) and causes Gambian or
Central African sleeping sickness (Fig. 7.2).
2. T. rhodesiense is transmitted by Glossina morsitans causing Rhodesian or East
African sleeping sickness.
3. T. cruzi is transmitted by a bug called Triatoma megista and causes Chagas disease or
American trypanosomiasis.
Kala – azar or visceral leishmaniasis is caused by Leishmania donovani, which is
transmitted by the vector Phlebotomus (sand fly). Infection may occur in the endothelial
cells, bone marrow, liver, lymph glands and blood vessels of the spleen. Symptoms of Kala
azar are weight loss, anaemia, fever, enlargement of spleen and liver.

Malaria is caused by different types of Plasmodium species such as P. vivax, P. ovale, P.
malariae and P. falciparum (Table 7.3). Plasmodium lives in the RBC of human in its
mature condition it is called as trophozoite. It is transmited from one person to another by
the bite of the infected female Anopheles mosquito.
Life cycle of Plasmodium

Plasmodium vivax is a digenic parasite, involving two hosts, man as the secondary host and
female Anopheles mosquito as the primary host. The life cycle of Plasmodium involves
three phases namely schizogony, gamogony and sporogony (Fig. 7.3).
The parasite first enters the human blood stream through the bite of an infected female
Anopheles mosquito. As it feeds, the mosquito injects the saliva containing the
sporozoites. The sporozoite within the blood stream immediately enters the hepatic cells of
the liver. Further in the liver they undergo multiple asexual fission (schizogony) and
produce merozoites. After being released from liver cells, the merozoites penetrate the
RBC’s.
Inside the RBC, the merozoite begins to develop as unicellular trophozoites. The
trophozoite grows in size and a central vacuole develops pushing them to one side of
cytoplasm and becomes the signet ring stage. The trophozoite nucleus then divides
asexually to produce the schizont. The large schizont shows yellowish - brown pigmented
granules called Schuffners granules. The schizont divides and produces mononucleated
merozoites. Eventually the erythrocyte lyses, releasing the merozoites and haemozoin toxin
into the blood stream to infect other erythrocytes. Lysis of red blood cells results in cycles
of fever and other symptoms. This erythrocytic stage is cyclic and repeats itself
approximately every 48 to 72 hours or longer depending on the species of Plasmodium
involved. The sudden release of merozoites triggers an attack on the RBCs. Occasionally,
merozoites differentiate into macrogametocytes and microgametocytes. When these are
ingested by a mosquito, they develop into male and female gametes respectively.
In the mosquito's gut, the infected erythrocytes lyse and male and female gametes fertilize
to form a diploid zygote called ookinete. The ookinete migrates to the mosquito's gut wall
and develop into an oocyte. The oocyte undergoes meiosis by a process called sporogony
to form sporozoites. These sporozoites migrate to the salivary glands of the mosquito. The
cycle is now completed and when the mosquito bites another human host, the sporozoites
are injected and the cycle begins a new.
The pathological changes caused by malaria, affects not only the erythrocytes but also the
spleen and other visceral organs. Incubation period of malaria is about 12 days. The early
symptoms of malaria are headache, nausea and muscular pain.
The classic symptoms first develop with the synchronized release of merozoites,
haemozoin toxin and erythrocyte debris into the blood stream resulting in malarial
paroxysms – shivering chills, high fever followed by sweating. Fever and chills are caused
partly by malarial toxins that induce macrophages to release tumour necrosis factor (TNF-
α) and interleukin.
Prevention
It is possible to break the transmission cycle by killing the insect vector. Mosquito's lay
their eggs in water. Larvae hatch and develop in water but breathe air by moving to the
surface. Oil can be sprayed over the water surface, to make it impossible for mosquito
larvae and pupae to breathe.
Ponds, drainage ditches and other permanent bodies of water can be stocked with fishes
such as Gambusia which feed on mosquito larvae. Preparations containing Bacillus
thuringiensis can be sprayed to kill the mosquito larvae since it is not toxic to other forms
of life. The best protection against malaria is to avoid being bitten by mosquito. People are
advised to use mosquito nets, wire gauging of windows and doors to prevent mosquito
bites.
In the 1950’s the World Health Organisation (WHO) introduced the Malaria eradication
programme. This programme was not successful due to the resistance of Plasmodium to the
drugs used to treat it and resistance of mosquito's to DDT and other insecticides.
drugs used to treat it and resistance of mosquito's to DDT and other insecticides.
3. Fungal diseases
Fungi was recognized as a causative agent of human diseases much earlier than bacteria.
Dermatomycosis is a cutaneous infection caused by fungi belonging to the genera
Trichophyton, Microsporum and Epidermophyton.
Ringworm is one of the most common fungal disease in humans (Fig. 7.4). Appearance of
dry, scaly lesions on the skin, nails and scalp are the main symptoms of the disease. Heat
and moisture help these fungi to grow and makes them to thrive in skin folds such as those
in the groin or between the toes. Ringworms of the feet is known as Athlete’s foot caused
by Tinea pedis (Fig. 7.5) . Ringworms are generally acquired from soil or by using clothes,
towels and comb used by infected persons.
4. Helminthic diseases
Helminthes are mostly endoparasitic in the gut and blood of human beings and cause
diseases called helminthiasis. The two most prevalent helminthic diseases are Ascariasis
and Filariasis.
Ascaris is a monogenic parasite and exhibits sexual dimorphism. Ascariasis is a disease
caused by the intestinal endoparasite Ascaris lumbricoides commonly called the round
worms (Fig. 7.6). It is transmitted through ingestion of embryonated eggs through
contaminated food and water. Children playing in contaminated soils are also prone to have
a chance of transfer of eggs from hand to mouth.

The symptoms of the disease are abdominal pain, vomiting, headache, anaemia, irritability
and diarrhoea. A heavy infection can cause nutritional deficiency and severe abdominal
pain and causes stunted growth in children. It may also cause enteritis, hepatitis and
bronchitis.
Filariasis is caused by Wuchereria bancrofti , commonly called filarial worm. It is found
in the lymph vessels and lymph nodes of man (Fig. 7.7). Wuchereria bancrofti is
sexually dimorphic, viviparous and digenic. The life cycle is completed in two hosts, man
and the female Culex mosquito The female filarial worm gives rise to juveniles called
microfilariae larvae. In the lymph glands, the juveniles develop into adults. The
accumulation of the worms block the lymphatic system resulting in inflammation of the
lymph nodes.

In some cases, the obstruction of lymph vessels causes elephantiasis or filariasis of the
limbs, scrotum and mammary glands (Fig. 7.8).
From <https://www.brainkart.com/article/Common-diseases-in-human-beings_38087/>

Organisms of health and agricultural importance

Scientific Names of Common Plants
Scientific Names of Common Plants
Common Name of Plants Scientific Name of Plants

Apple Pyrus malus
Bamboo Bamboosa aridinarifolia
Brinjal Solanum melongena
Banana Musa paradisicum
Black Gram Palsoes mungo
Banyan Ficus benghalensis
Barley Hordeum vulgare
Black Pepper Piper nigrum
Carrot Daucas carota
Cashew nut Anacardium occidentale
Clove Syzygium aromaticum
Coriander Coriandrum sativum
Cucumber Cucumis sativas
Curry leaf Murraya koenigii
Capsicum Capsicum fruitscence
Chiku Achras sapota
Cotton Gossypium herbaceum
Dragon fruit Hylocereus undatus
Finger millet Eleusine coracana
Green Gram Phaseolus audicus
Guava Psidium guava
Ginger Zingiber officinale
Garlic Allium sativum
Jack fruit Artocarpus integra
Jowar Sorghum Vulgare
Kadamb Anthocephalus indicus
Lemon Citrus Limonium
Maize Zea mays
Mango Mangifera indica
Neem Azadirachta indica
Onion Allium cepa
Orange Citrus aurantium
Pea Pisum sativum
Papaya Carica papaya
Potato Solanum tuberosum

Potato Solanum tuberosum
Pomegranate Punica granatum
Peacock Flower (Gulmohar) Delonix regia rafin
Purple orchid tree (Kachnar) Bauhinia purpurea
Peepal Ficus religiosa Linn.
Pineapple Ananus sativus
Radish Raphanus sativus
Red maple Acer rubrum
Rice Oryza sativa
Rose Rosa
Soya bean Glycine max
Silver Oak Grevillea robusta
Sandalwood Santalum album
Spinach Lactuca sativa
Sunflower Helianthus annuus
Turmeric Curcuma longa
Tobacco Nicotina tobaccum
Tulsi Ocimum sanctum
Teak Tectona grandis Linn.
Tamarind tree Tamarindus indica
Tomato Lycopersicon esculentum
Watermelon Citrullus vulgaris
Lettuce Lactuca sativa
Mint Mentha arvensis
Binomial Nomenclature for Animals

The naming system for animals is similar to the naming system of plants. While writing a
scientific name, the genus comes first followed by the species. Except, Scientific name of
plants cannot have similar two parts of the binomial name. While in Animals, this is
allowed. Example- Scientific name of American Bison is- Bison bison.
Scientific Names of Common Animals
Common Name of Animals Scientific Name of Animals

Dog Canis lupus
Domestic dog Canis lupus familiaris
Cat Felis catus
Horse Equus ferus caballus
Sheep Ovis aries
Sheep Ovis aries
Domestic Pig Sus scrofa domesticus
Goat Capra aegagrus hircus
Donkey Equus africanus asinus
Dromedary Camel Camelus dromedaries
Bactrian Camel Camelus bactrianus
Water Buffalo Bubalus bubalis
Cow Bos Taurus
Indian Elephant Elephas maximus
Tiger Panthera tigris
Lion Felis leo
Fowl Gallus gallus domesticus
Blackbuck Antilope cervicapra
Bulbul Molpastes cafer
Cheetah Acinonyx jubatus
Red fox Vulpes Vulpes
Gharial Gavialis gangeticus

Hippopotamus Hippopotamus amphibius
House wall Lizard Hemidactylus flaviviridis
Indian Cobra Naja naja
King cobra Ophiophagus hannah

Rat snake Ptyas mucosa
Scientific Names of Extinct Animals
Passenger pigeon Ectopistes migratorius

Tasmanian tiger Thylacinus cynocephalus
Moa Dinornithiformes
T-Rex Tyrannosaurus rex
Dolphin Lipotes vexillifer
Tasmanian Tiger Thylacinus cynocephalus
From <https://www.vedantu.com/biology/scientific-names-of-plants-and-animals>

Levels of Organization in Multicellular Organisms
Sunday, 31 October, 2021 10:45 AM
The simplest living multicellular organisms, sponges, are made of many specialized types of cells that work together for a common goal. Such cell types
include digestive cells, tubular pore cells; and epidermal cells. Though the different cell types create a large organized, multicellular structure—the visible
sponge—they are not organized into true interconnected tissues. If a sponge is broken up by passing it through a sieve, the sponge will reform on the other
side. However, if the sponge’s cells are separated from each other, the individual cell types cannot survive alone. Simpler colonial organisms, such as members
of the genus Volvox, differ in that their individual cells are free-living and can survive on their own if separated from the colony.
A tissue is a group of connected cells that have a similar function within an organism.More complex organisms such as jellyfish, coral, and sea anemones have
a tissue level of organization. For example, jellyfish have tissues that have separate protective, digestive, and sensory functions. Though most animals have
many different types of cells, they only have four basic types of tissue: connective, muscle, nervous, and epithelial.
Even more complex organisms, such as the roundworm, while also having differentiated cells and tissues, have an organ level of development. An organ is a
group of tissues that has a specific function or group of functions. Organs can be as primitive as the brain of a flatworm (a group of nerve cells), as large as the
stem of a sequoia (up to 90 meters, or 300 feet, in height), or as complex as a human liver.
The most complex organisms (such as mammals, trees, and flowers) have organ systems. An organ system is a group of organs that act together to carry out
complex related functions, with each organ focusing on a part of the task. An example is the human digestive system in which the mouth ingests food, the
stomach crushes and liquifies it, the pancreas and gall bladder make and release digestive enzymes, and the intestines absorb nutrients into the blood.
From <https://www.ck12.org/book/ck-12-biology-advanced-concepts/section/3.24/>
Animal Tissues
• Blood and muscles are both examples of tissues found in our body. On the basis of the functions they perform we can think of different types of animal
tissues, such as epithelial tissue, connective tissue, muscular tissue and nervous tissue.
• Blood is a type of connective tissue, and muscle forms muscular tissue.
Epithelial Tissue
• The covering or protective tissues in the animal body are epithelial tissues.
• Epithelium covers most organs and cavities within the body.
• It also forms a barrier to keep different body systems separate.
• The skin, the lining of the mouth, the lining of blood vessels, lung alveoli and kidney tubules are all made of epithelial tissue.
• Epithelial tissue cells are tightly packed and form a continuous sheet.
• They have only a small amount of cementing material between them and almost no intercellular spaces.
• Obviously, anything entering or leaving the body must cross at least one layer of epithelium.
• As a result cells of various epithelia play an important role in regulating the exchange of materials between the body and the external environment and
also between different parts of the body.
• Regardless of the type, all epithelium is usually separated from the underlying tissue by an extracellular fibrous basement membrane.
• There are two types of epithelial tissues namely simple epithelium and compound epithelium.
Simple Epithelium
• Simple epithelium is composed of a single layer of cells and functions as a lining for body cavities, ducts, and tubes.
Squamous Epithelium
• The squamous epithelium is made of a single thin layer of flattened cells with irregular boundaries.
• They are found in the walls of blood vessels and air sacs of lungs and are involved in functions like forming a diffusion boundary.
Levels of Organization Page 120

• They are found in the walls of blood vessels and air sacs of lungs and are involved in functions like forming a diffusion boundary.
• Different epithelia show differing structures that correlate with their unique functions. For example, in cells lining blood vessels or lung alveoli, where
transportation of substances occurs through a selectively Permeable surface, there is a simple flat kind of epithelium. This is called the simple
Squamous epithelium.
• Simple squamous epithelial cells are extremely thin and flat and form a delicate lining.
• The oesophagus and the lining of the mouth are also covered with squamous epithelium.
Stratified Squamous Epithelium
• The skin, which protects the body, is made of squamous epithelium.
• Skin epithelial cells are arranged in many layers to prevent wear and tear.
• Since they are arranged in a pattern of layers, the epithelium is called stratified squamous epithelium.
Ciliated Columnar Epithelium
• The columnar epithelium is composed of a single layer of tall and slender cells. Their nuclei are located at the base.
• Where absorption and secretion occur, as in the inner lining of the intestine, tall epithelial cells are present.
• In the respiratory tract, the columnar epithelial tissue also has cilia, which are hair-like projections on the outer surfaces of epithelial cells. These cilia
can move, and their movement pushes the mucus forward to clear it. This type of epithelium is thus ciliated columnar epithelium.
• They are mainly present in the inner surface of hollow organs like bronchioles and fallopian tubes.
Cuboidal Epithelium
• The cuboidal epithelium is composed of a single layer of cube-like cells. This is commonly found in ducts of glands and tubular parts of nephrons in
kidneys and its main functions are secretion and absorption.
• Cuboidal epithelium (with cube-shaped cells) forms the lining of kidney tubules and ducts of salivary glands, where it provides mechanical support.
Glandular Epithelium
• Epithelial cells often acquire additional specialization as gland cells, which can secrete substances at the epithelial surface.
• Sometimes a portion of the epithelial tissue folds inward, and a multicellular gland is formed. This is glandular epithelium.
• Some of the columnar or cuboidal cells get specialized for secretion and are called glandular epithelium. They are mainly of two types: unicellular,
consisting of isolated glandular cells (goblet cells of the alimentary canal), and multicellular, consisting of cluster of cells (salivary gland).
• On the basis of the mode of pouring of their secretions, glands are divided into two categories namely EXOCRINE and ENDOCRINE.
• Exocrine glands secrete mucus, saliva, earwax, oil, milk, digestive enzymes and other cell products. These products are released through ducts or tubes.
• In contrast, endocrine glands do not have ducts. Their products called hormones are secreted directly into the fluid bathing the gland.
Compound Epithelium
• The compound epithelium consists of two or more cell layers and has protective function as it does in our skin.
• Compound epithelium is made of more than one layer (multi-layered) of cells and thus has a limited role in secretion and absorption. Their main
function is to provide protection against chemical and mechanical stresses.
• They cover the dry surface of the skin, the moist surface of buccal cavity, pharynx, inner lining of ducts of salivary glands and of pancreatic ducts.
• All cells in epithelium are held together with little intercellular material. In nearly all animal tissues, specialized junctions provide both structural and
functional links between its individual cells.
• Three types of cell junctions are found in the epithelium and other tissues. These are called as tight, adhering and gap junctions.
• Tight junctions help to stop substances from leaking across a tissue. Adhering junctions perform cementing to keep neighboring cells together. Gap
junctions facilitate the cells to communicate with each other by connecting the cytoplasm of adjoining cells, for rapid transfer of ions, small molecules
and sometimes big molecules.
Connective Tissue
• Connective tissues are most abundant and widely distributed in the body of complex animals. They are named connective tissues because of their
special function of linking and supporting other tissues/organs of the body.
• They range from soft connective tissues to specialized types, which include cartilage, bone, adipose, and blood.
• In all connective tissues except blood, the cells secrete fibres of structural proteins called collagen or elastin.
• The fibres provide strength, elasticity and flexibility to the tissue. These cells also secrete modified polysaccharides, which accumulate between cells
and fibres and act as matrix (ground substance).
• Connective tissues are classified into three types: (i) Loose connective tissue, (ii) Dense connective tissue and (iii) Specialized connective tissue.
Loose Connective Tissue
• Loose connective tissue has cells and fibres loosely arranged in a semi-fluid ground substance, for example, areolar tissue present beneath the skin.
• Often it serves as a support framework for epithelium. It contains fibroblasts (cells that produce and secrete fibres), macrophages [a large phagocytic
cell found in stationary form in the tissues or as a mobile white blood cell, especially at sites of infection] and mast cells [a cell found in connective
tissue and releasing histamine and other substances during inflammatory and allergic reactions].
• Adipose tissue is a type of loose connective tissue located mainly beneath the skin. The cells of this tissue are specialized to store fats. The excess of
nutrients which are not used immediately are converted into fats and are stored in this tissue.
Dense Connective Tissue
• Fibres and fibroblasts are compactly packed in the dense connective tissues. Orientation of fibres show a regular or irregular pattern and are called
dense regular and dense irregular tissues.
• In the dense regular connective tissues, the collagen fibres are present in rows between many parallel bundles of fibres. Tendons, which attach skeletal
muscles to bones and ligaments which attach one bone to another are examples of this tissue.
• Dense irregular connective tissue has fibroblasts and many fibres (mostly collagen) that are oriented differently. This tissue is present in the skin.
Specialized Connective Tissue – Cartilage, Bones, Blood, Areolar
• Cartilage, bones and blood are various types of specialized connective tissues.
• The intercellular material of cartilage is solid and pliable and resists compression. Cells of this tissue (chondrocytes) are enclosed in small cavities within
the matrix secreted by them.
• Most of the cartilages in vertebrate embryos are replaced by bones in adults. Cartilage is present in the tip of nose, outer ear joints, trachea, larynx,
between adjacent bones of the vertebral column, limbs and hands in adults.
• Bone cells are embedded in a hard matrix that is composed of calcium and phosphorus compounds.
• Bones have a hard and non-pliable ground substance rich in calcium salts and collagen fibres which give bone its strength. It is the main tissue that

• Bones have a hard and non-pliable ground substance rich in calcium salts and collagen fibres which give bone its strength. It is the main tissue that
provides structural frame to the body. Bones support and protect softer tissues and organs.
• The bone cells (osteocytes) are present in the spaces called lacunae. The bone marrow in some bones is the site of production of blood cells.
• Two bones can be connected to each other by another type of connective tissue called the ligament. This tissue is very elastic. It has considerable
strength. Ligaments contain very little matrix. Tendons connect bones to muscles and are another type of connective tissue. Tendons are fibrous tissue
with great strength but limited flexibility.
• Blood is a fluid connective tissue containing plasma, red blood cells (RBC), white blood cells (WBC) and platelets. It is the main circulating fluid that
helps in the transport of various substances.
• Areolar connective tissue is found between the skin and muscles, around blood vessels and nerves and in the bone marrow.
• It fills the space inside the organs, supports internal organs and helps in repair of tissues.
Muscular Tissue
• Each muscle is made of many long, cylindrical fibres arranged in parallel arrays. These fibres are composed of numerous fine fibrils, called myofibrils.
• Muscle fibres contract (shorten) in response to stimulation, then relax (lengthen) and return to their uncontracted state in a coordinated fashion.
Muscles contain special proteins called contractile proteins, which contract and relax to cause movement.
• Muscles are of three types, skeletal, smooth, and cardiac.
Skeletal Muscle Tissue – Voluntary Muscles

• We can move some muscles by conscious will. Such muscles are called voluntary muscles.
• These muscles are also called skeletal muscles as they are mostly attached to bones and help in body movement.
• Under the microscope, these muscles show alternate light and dark bands or striations. As a result, they are also called striated muscles. The cells of this
tissue are long, cylindrical, unbranched and multinucleate (having many nuclei).
• Skeletal muscle tissue is closely attached to skeletal bones. In a typical muscle such as the biceps, striated (striped) skeletal muscle fibres are bundled
together in a parallel fashion. A sheath of tough connective tissue encloses several bundles of such muscle fibres.
Smooth Muscle Tissue – Involuntary Muscles
• The movement of food in the alimentary canal or the contraction and relaxation of blood vessels are involuntary movements. Wecannot really start
them or stop them simply by wanting to do so! Smooth muscles or involuntary muscles control such movements.
• They are also found in the iris of the eye, in ureters and in the bronchi of the lungs.
• The cells are long with pointed ends (spindle-shaped) and uninucleate (having a single nucleus). They are also called unstriated muscles.
• The smooth muscle fibres taper at both ends (fusiform, spindle-shaped) and do not show striations. Cell junctions hold them together and they are
bundled together in a connective tissue sheath. The wall of internal organs such as the blood vessels, stomach and intestine contains this type of muscle
tissue.
Cardiac Muscle Tissue – Involuntary Muscles
• The muscles of the heart show rhythmic contraction and relaxation throughout life. These involuntary muscles are called cardiac muscles. Heart muscle
cells are cylindrical, branched and uninucleate.
• Cardiac muscle tissue is a contractile tissue present only in the heart. Cell junctions fuse the plasma membranes of cardiac muscle cells and make them
stick together. Communication junctions (intercalated discs) at some fusion points allow the cells to contract as a unit, i.e., when one cell receives a
signal to contract, its neighbors are also stimulated to contract.
Nervous Tissue
• Neural tissue exerts the greatest control over the body’s responsiveness to changing conditions.
• Neurons, the unit of neural system are excitable cells. The neuroglial cell which constitute the rest of the neural system protect and support neurons.
• Neuroglia make up more than one-half the volume of neural tissue in our body.
• When a neuron is suitably stimulated, an electrical disturbance is generated which swiftly travels along its plasma membrane.
• Arrival of the disturbance at the neuron’s endings, or output zone, triggers events that may cause stimulation or inhibition of adjacent neurons and
other cells.
• All cells possess the ability to respond to stimuli. However, cells of the nervous tissue are highly specialized for being stimulated and then transmitting
the stimulus very rapidly from one place to another within the body.
• The brain, spinal cord and nerves are all composed of the nervous tissue. The cells of this tissue are called nerve cells or neurons.
• A neuron consists of a cell body with a nucleus and cytoplasm, from which long thin hair-like parts arise. Usually each neuron has a single long part,
called the axon, and many short, branched parts called dendrites.
• An individual nerve cell may be up to a metre long. Many nerve fibres bound together by connective tissue make up a nerve.
• Nerve impulses allow us to move our muscles when we want to. The functional Combination of nerve and muscle tissue is fundamental to most animals.
This combination enables animals to move rapidly in response to stimuli.

From <https://www.pmfias.com/animal-tissues-epithelium-tissue-connective-tissue-muscular-tissue-nervous-tissue/>

Levels of Structural Organization

Biodiversity in India
Environment
Biodiversity of India, Biodiversity Hotspots of India
• According to IUCN (2004), the total number of plant and animal species described so far is
slightly more than 1.5 million.
• Estimates place the global species diversity at several million.
• A large proportion of the species waiting to be discovered are in the tropics.
• More than 70 per cent of all the species recorded are animals, while plants (including algae,
fungi, bryophytes, gymnosperms and angiosperms) comprise no more than 22 per cent of the
total.
• Among animals, insects are the most species-rich taxonomic group, making up more than 70
per cent of the total.
• The number of fungi species in the world is more than the combined total of the species of
fishes, amphibians, reptiles and mammals.
• The largely tropical Amazonian rain forest in South America has the greatest biodiversity on
earth.
Definitions
Biodiversity
• Biodiversity is the variety of plant and animal life in the world or in a particular habitat.
• Biodiversity is measured by two major components: species richness, and species evenness.
Species richness
• It is the measure of the number of species found in a community.
Species evenness
• Species evenness is a measure of the relative abundance of the different species making up
the richness of an area.
• Example: The sample forest A has 4 tigers, 5 deer and 6 rabbits and sample forest B has 1 tiger,
6 deer and 8 rabbits. Both samples have the same richness (3 species – species richness) and
the same total number of individuals (15). However, the sample forest A has more evenness
than the sample forest B.
• Low evenness indicates that a few species dominate the site.
Alpha diversity
• It refers to the diversity within a particular area or ecosystem and is usually expressed by the
number of species (i.e., species richness) in that ecosystem.
Beta diversity
• It is a comparison of diversity between ecosystems, usually measured as the change in the
amount of species between the ecosystems.
Gamma diversity
• It is a measure of the overall diversity for the different ecosystems within a region.
Genetic diversity
• Genetic diversity is the total number of genetic characteristics in the genetic makeup of a
species.
• A single species might show high diversity at the genetic level (E.g. Homo sapiens: Chinese,
Indian American, African etc.).
• India has more than 50,000 genetically different strains of rice and 1,000 varieties of mango.
• Genetic diversity allows species to adapt to changing environments. This diversity aims to
ensure that some species survive drastic changes and thus carry on desirable genes.
• Species that differ from one another in their genetic makeup do not interbreed in nature.
• Closely-related species have in common much of their hereditary characteristics. For instance,
about 98.4 per cent of the genes of humans and chimpanzees are the same.
Species diversity
• It is the ratio of one species population over total number of organisms across all species in
the given biome. ‘Zero’ would be infinite diversity, and ‘one’ represents only one species
present.
• Species diversity is a measure of the diversity within an ecological community that
incorporates both species richness (the number of species in a community) and the evenness
of species.
• In general, species diversity decreases as we move away from the equator towards the poles.
• With very few exceptions, tropics (latitudinal range of 23.5° N to 23.5° S) harbour more species
than temperate or polar areas.
• Bioprospecting: nations endowed with rich biodiversity explore molecular, genetic and species-
level diversity to derive products of economic importance.
Stable community
• A stable community means that there is not much variation in productivity from year to year ;
it is either resistant or resilient to occasional disturbances (natural or human-made) and is
resistant to invasions by alien species.
Ecological diversity
• Ecological diversity refers to different types of habitats. A habitat is the cumulative factor of
the climate, vegetation and geography of a region.
• It includes various biological zones, like a lake, desert, coast, estuaries, wetlands, mangroves,
coral reefs etc.
• At the ecosystem level, India, for instance, with its deserts, rain forests, mangroves, coral
reefs, wetlands, estuaries, and alpine meadows has a greater ecosystem diversity than a
Scandinavian country like Norway.
Endemism
Biodiversity Page 126

Endemism
• There are more than 200000 species in India of which several are confined to India (endemic).
• Endemism is the ecological state of a species being unique to a defined geographic location ,
such as an island, nation, country or other defined zone, or habitat type; organisms that are
indigenous to a place are not endemic to it if they are also found elsewhere.
• A particular type of animal or plant may be endemic to a zone, a state or a country. The
extreme opposite of endemism is cosmopolitan distribution.
Keystone species
• Keystone speciesis a species whose addition to or loss from an ecosystem leads to major
changes in the occurrence of at least one other species .
• Certain species in an ecosystem is considered more important in determining the presence of
many other species in that ecosystem.
• All top predators (Tiger, Lion, Crocodile, Elephant) are considered as keystone species
because they regulate all other animal population indirectly.
• Hence top predators are given much consideration in conservation.
• If keystone species is lost, it will result in the degradation of the whole ecosystem.
• For example, certain plant species (ebony tree, Indian-laurel) exclusively depends upon bats
for its pollination. If the bat population is reduced, then regeneration of particular plants
becomes more difficult.
Foundation species
• Foundation species is a dominant primary producerin an ecosystem both in terms of
abundance and influence. Example: kelp in kelp forests and corals in coral reefs.
Flagship species
• A flagship species is a species chosen to represent an environmental cause, such as an
ecosystem in need of conservation.
• These species are chosen for their vulnerability, attractiveness or distinctiveness in order to
engender support and acknowledgement from the public at large.
• Example: Indian tiger, African elephant, giant panda of China, the leatherback sea turtle, etc.
Biodiversity of India
• India is recognized as one of the mega-diverse countries, rich in biodiversity and associated
traditional knowledge.
• India has 23.39% of its geographical area under forest and tree cover.
• With just 2.4% of the land area, India accounts for nearly 7% of the recorded species even
while supporting almost 18% of the human population.
• In terms of species richness, India ranks seventh in mammals, ninth in birds and fifth in
reptiles.
• In terms of endemism of vertebrate groups, India’s position is tenth in birds with 69
species, fifth in reptiles with 156 species and seventh in amphibians with 110 species.
• India’s share of crops is 44% as compared to the world average of 11%.
India Represents
• Two ‘Realms’
• Five Biomes
• Ten Bio-geographic Zones
• Twenty-five Bio-geographic provinces
Realms
• Biogeographic realms are large spatial regions within which ecosystems share a broadly
similar biota.
• A realm is a continent or sub-continent sized area with unifying features of geography and
fauna & flora.
• The Indian region is composed of two realms. They are:
1. the Himalayan region represented by Palearctic Realm and
2. the rest of the sub-continent represented by Malayan Realm
• In world, Eight terrestrial biogeographic realms are typically recognised. They are
1. Nearctic Realm
2. Palaearctic Realm
3. Africotropical Realm
4. Indomalayan Realm
5. Ocenaia Realm
6. Australian Realm
7. Antarctic Realm
8. Neotropical Realm
Biomes of India
• The term biome means the main groups of plants and animals living in areas of certain
climate patterns.
• It includes the way in which animals, vegetation and soil interact together. The plants and
animals of that area have adapted to that environment.
The five biomes of India are:
1. Tropical Humid Forests
2. Tropical Dry or Deciduous Forests (including Monsoon Forests)
3. Warm deserts and semi-deserts
4. Coniferous forests and
5. Alpine meadows.
Bio-geographic Zones
• Biogeography deals with the geographical distribution of plants and animals .
• Biogeographic zones were used as a basis for planning wildlife protected areas in India.
• There are 10 biogeographic zones which are distinguished clearly in India. They are as follows:
1. Trans-Himalayas
2. Himalayas
3. Desert
4. Semi-arid
5. Western Ghats
6. Deccan Peninsula
7. Gangetic plain
8. North-east India
9. Islands
10. Coasts
Bio-geographic provinces
• Bio-geographic Province is an ecosystematic or biotic subdivision of realms.
• India is divided into 25 bio geographic zones.
Biogeographic Zones (10) Biogeographic Provinces (25)
1. Trans Himalaya 1. 1A: Himalaya – Ladakh Mountains
2. 1B: Himalaya – Tibetan Plateau
3. 1C: Trans – Himalaya Sikkim
1. The Himalaya 1. 2A: Himalaya – North West Himalaya
2. 2B: Himalaya – West Himalaya
3. 2C: Himalaya – Central Himalaya
4. 2D: Himalaya – East Himalaya

4. 2D: Himalaya – East Himalaya
1. The Indian Desert 1. 3A: Desert – Thar
2. 3B: Desert – Kutch
1. The Semi-Arid 1. 4A: Semi-Arid – Punjab Plains
2. 4B: Semi-Arid – Gujarat Rajputana
1. The Western Ghats 1. 5A: Western Ghats – Malabar Plains
2. 5B: Western Ghats – Western Ghats Mountains
1. The Deccan Peninsula 1. 6A: Deccan Peninsular – Central Highlands
2. 6B: Deccan Peninsular – Chotta Nagpur
3. 6C: Deccan Peninsular – Eastern Highlands
4. 6D: Deccan Peninsular – Central Plateau
5. 6E: Deccan Peninsular – Deccan South
1. The Gangetic Plains 1. 7A: Gangetic Plain – Upper Gangetic Plains
2. 7B: Gangetic Plain – Lower Gangetic Plains
1. The Coasts 1. 8A: Coasts – West Coast
2. 8B: Coasts – East Coast
3. 8C: Coasts – Lakshadweep
1. Northeast India 1. 9A: North-East – Brahmaputra Valley
2. 9B: North-East – North East Hills
1. Islands 1. 10A: Islands – Andaman
2. 10B: Islands – Nicobars
Wildlife Diversity of India

Himalayan mountain system
• The west Himalayas have low rainfall, heavy snowfall (temperate conditions).
• In the east Himalayas, there is heavy rainfall, snowfall only at very high altitudes.
• Lower altitudes conditions are similar to the tropical rain forests.
Himalayan foothills
• Flora: Natural monsoon evergreen and semi-evergreen forests; dominant species are sal, silk-
cotton trees, giant bamboos; tall grassy meadow with savannahs in terai .
• Fauna: Elephant, sambar, swamp deer, cheetal, hog deer, barking deer, wild boar tiger,
panther, hyena, black bear, sloth bear, Great Indian one -horned rhinoceros, wild buffalo,
Gangetic gharial, golden langur.
Western Himalayas (High altitude region)
• Flora: Natural monsoon evergreen and semi-evergreen forests; rhododendrons; dwarf hill
bamboo and birch forests mixed with alpine pastures .
• Fauna: Tibetan wild ass (kiang) (Don’t confuse this with Asiatic wild ass which in found in
Kutch region), wild goats (thar, ibex) and blue sheep; antelopes (Chiru and Tibetan gazelle),
deers (hangul of Kashmir stag and shou or Sikkim stag, musk deer); golden eagle, snow
cocks, snow partridges; snow leopard, black and brown bears; birds like Griffon vultures.
Q. What is the difference between the antelopes Oryx and Chiru?
1. Oryx is adapted to live in hot and arid areas whereas Chiru is adapted to live in steppes and
semi-desert areas of cold high mountains.
2. Oryx is poached for its antlers whereas Chiru is poached for its musk.
3. Oryx exists in western India only whereas Chiru exists in north-east India only.
4. None of the statements a, b, and c given above is correct.
• They are both antelopes.
Answer: a)
Eastern Himalayas
• Flora: Oaks, magnolias, laurels and birches covered with moss and ferns; coniferous forests of
pine, fir, yew and junipers with an undergrowth of scrubby rhododendrons and dwarf
bamboos; lichens, mosses, orchids, and other epiphytes dominant (due to high humidity and
high rainfall).
• Fauna: Red panda, hog badgers, forest badgers, crestless porcupines, takins etc.
Peninsular – Indian sub-region

• It has two zones.
1. peninsular India and its extension into the drainage basin of the Ganges river system, and
2. desert region of Rajasthan-the Thar of Indian desert region.
Peninsular India
• It is home to tropical moist deciduous to tropical dry deciduous and scrub vegetation
depending upon the variation in rainfall and humidity.
• Flora: Sal in north and east extensions (higher rainfall) and teak in southern plateau are
dominant trees.
• West Ghats have evergreen vegetation (flora and fauna similar to evergreen rainforests of
northeastern of India. In dry areas of Rajasthan and Aravalli hills, trees are scattered, and
thorny scrub species predominate. The forests give way to more open savannah habit.
• Fauna: Elephant, wild boar, deers (cheetal or axis deer), hog deer swamp deer or barasinga,
sambar, muntjak or barking deer, antelopes (four-horned antelope, Nilgiri, blackbuck,
chinkara gazelle), wild dog or dhole, tiger, leopard, cheetah, lion, wild pig, monkey, striped
hyena, jackal, gaur.
Indian desert
• Thar desert of Rajasthan has unique flora and fauna.
• Flora: Thorny trees with reduced leaves; cacti, other succulents are the main plants.
• Fauna: Animals are mostly burrowing ones. Among mammals’ rodents are the largest group.
• The Indian desert gerbils are mouse-like, rodents, other animals are, Asiatic wild ass, black
buck, desert cat, caracal, red fox; reptiles (snakes, lizards and tortoise) well represented.
• Desert lizards include agamids and geckos. Among birds, the most discussed is Great Indian
Bustard.
Tropical rain forest region

• Distributed in areas of Western Ghats and northeast India.
• Flora: Extensive grasslands interspersed with densely forested gorges of evergreen vegetation
known as sholas occur in the Nilgiris (an offshoot of Western Ghats). Sholas also occur in
Annamalai and Palani hills.
• The rain forests of the Western Ghats have dense and lofty trees with much species diversity.

• The rain forests of the Western Ghats have dense and lofty trees with much species diversity.
Mosses, ferns, epiphytes, orchids, lianas and vines, herbs, shrubs make diverse
habitat. Ebony trees predominate in these forests.
• Fauna: It is very rich with all kinds of animals. There are wild elephants, gaur and other larger
animals.
• Most species are tree dwellers. The most prominent are hoolock gibbon (only ape found in
India), golden langur, capped langur or leaf monkey, Assam macaque and the pig -tailed
macaque, lion-tailed macaque, Nilgiri langur slender loris, bats, giant squirrel, civets, flying
squirrels, Nilgiri mongoose, spiny mouse.
Andaman and Nicobar Islands

• Flora: These are home for tropical rain forests. Mangroves are distributed in the coastal areas.
• Fauna: Among mammals, bats and rats; Andaman pig, crab-eating macaque, palm civet and
deers (spotted deer, barking deer, hog deer, sambar).
• Among marine mammals, there are dugong, false killer whale, dolphin.
• Among birds are rare one is Narcondum hornbill, white-bellied sea-eagle.
• Salt-water crocodile, a number of marine turtles, coconut crab, lizards (the largest being
water monitor), 40 species of snakes including cobra, viper, voral and sea snake, python, etc.
are present.
Mangrove swamps of Sundarbans

• Sunderbans are the delta of the Ganges where both the Brahmaputra and the Ganges join and
drain into the Bay of Bengal.
• Flora: Various species of mangroves.
• Fauna. In the higher regions of mangroves, there are spotted deer, pigs, monitor lizard,
monkeys. The most interesting animal of Sunderbans is the Royal Bengal Tiger.
Biodiversity Hotspots
• Biodiversity hotspots are regions with high species richness and a high degree of endemism.
• The British biologist Norman Myers coined the term “biodiversity hotspot” in 1988 as a
biogeographic region characterized both by exceptional levels of plant endemism and by
serious levels of habitat loss.
• Conservation International (CI)adopted Myers’ hotspots and in 1996, the organization made
the decision to undertake a reassessment of the hotspots concept.
• According to CI, to qualify as a hotspot a region must meet two strict criteria:
1. It must contain at least 1,500 species of vascular plants (> 0.5% of the world’s total) as
endemics– which is to say, it must have a high percentage of plant life found nowhere
else on the planet. A hotspot, in other words, is irreplaceable.
2. It has to have lost at least 70% of its original habitat. (It must have 30% or less of its
original natural vegetation).In other words, it must be threatened.
• In 1999, CI identified 25 biodiversity hotspots in the book “Hotspots: Earth’s Biologically
Richest and Most Endangered Terrestrial Ecoregions”.
• In 2005 CI published an updated titled “Hotspots Revisited: Earth’s Biologically Richest and
Most Endangered Terrestrial Ecoregions”.
• The 35 biodiversity hotspots cover 2.3% of the Earth’s land surface, yet more than 50% of the
world’s plant species and 42% of all terrestrial vertebrate species are endemic to these areas.
• In 2011, the Forests of East Australia region was identified as the 35th biodiversity hotspot.
Biodiversity hotspots in India

1. Himalaya: Includes the entire Indian Himalayan region (and that falling in Pakistan,
Tibet, Nepal, Bhutan, China and Myanmar).
2. Indo-Burma: Includes entire North-eastern India, except Assam and Andaman group of
Islands (and Myanmar, Thailand, Vietnam, Laos, Cambodia and southern China)
3. Western Ghats and Sri Lanka: Includes entire Western Ghats (and Sri Lanka).
4. Sundalands: Includes Nicobar group of Islands (and Indonesia, Malaysia, Singapore,
Brunei, Philippines).

Eastern Himalayas, which was originally part of the Indo-Burma Biodiversity Hotspot and
included Bhutan, north-eastern India and southern, central and eastern Nepal.
In 2004, a hotspot reappraisal classified the region as part of two hotspots: Indo-Burma and
the newly distinguished Himalaya.
List of Biodiversity hotspots in India given in Geography notes must be ignored. The info
given here is the most accurate.
Source
Q. Consider the following statements: [2010]
1. Biodiversity hotspots are located only in tropical regions.
2. India has four biodiversity hotspots, i.e., Eastern Himalayas, Western Himalayas, Western
Ghats and Andaman and Nicobar Islands.
Which of the statements given above is/are correct?
1. 1 only
2. 2 only
3. Both 1 and 2
4. Neither I nor 2
Answer: d) neither
• The Himalaya Hotspot is home to important populations of numerous large birds and
mammals, including vultures, tigers, elephants, rhinos and wild water buffalo.
• Indo-Burma holds remarkable endemism in freshwater turtle species, most of which are
threatened with extinction, due to over-harvesting and extensive habitat loss.
• The spectacular flora and fauna of the Sundaland Hotspot are succumbing to the explosive
growth of industrial forestry in these islands and to the international animal trade that claims
tigers, monkeys, etc.
• Faced with tremendous population pressure, the forests of the Western Ghats and Sri Lanka
have been dramatically impacted by the demands for timber and agricultural land.
• The region also houses important populations of Asian Elephants, Indian Tigers, the Lion-
tailed Macaque, Niligiri tahr, Indian Giant squirrel , etc.
World Heritage Sites

• World Heritage Sites means “Sites any of various areas or objects inscribed on the United
Nations Educational, Scientific, and Cultural Organization (UNESCO) World Heritage List”.
• The sites are designated as having outstanding universal value under the Convention
concerning the Protection of the World Cultural and Natural Heritage .
• This Convention, which was adopted by the UNESCO in 1972 (and enforced in 1975) provides a
framework for international cooperation in preserving and protecting cultural treasures and
natural areas throughout the world. The first list of World Heritage state was published in
1978.
• The convention defines the kind of sites which can be considered for inscription of the World
heritage list (ancient monuments, museums, biodiversity and geological heritage,), and sets
out the duties of the State Parties in identifying potential sites and their role in protecting
them.
“Natural heritage sites are restricted to those natural areas that
1. furnish outstanding examples of the Earth’s record of life or its geologic processes.
2. provide excellent examples of ongoing ecological and biological evolutionary processes.
3. contain natural phenomena that are rare, unique, superlative, or of outstanding beauty
or
1. furnish habitats or rare endangered animals or plants or are sites of exceptional biodiversity”.
2. There are ten criteria for cultural heritage and natural heritage.
3. Nominated sites must be of “outstanding universal value” and meet at least one of the criteria
below.
International Year of Biodiversity
• The United Nations declared 2010 to be the International Year of Biodiversity.
• It is a celebration of life on earth and of the value of biodiversity for our lives.
Slogan
“Biodiversity is variety of life on earth
Biodiversity is life.
Biodiversity is our life”.
Man and Biosphere Programme (MAB programme)

• It was first started by UNESCO in 1971.
• Later introduced in India in 1986.
Aim
• Studying the effects of human interference and pollution on the biotic and abiotic components
of ecosystems.
• Conservation the ecosystems for the present as well as future.
The main objects of MAB programme are to:
• Conserve representative samples of ecosystem.
• Provide long term in situ conservation of genetic diversity.
• Provide opportunities for education and training.
• Provide appropriate sustainable managements of the living resources.
• Promote infer national co-operation.
From <https://www.pmfias.com/biodiversity-hotspots-india/>

Natural History of Indian Subcontinent

Organisms of Conservation Concern
Rare Species List

Mensuration Formulas
Monday, 6 December, 2021 04:41 PM
Mensuration Formulas For 2D Shapes

Shape Area (Square units) Perimeter (units) Figure
Square a2 4a
Rectangle l×b 2 ( l + b)
Circle πr2 2πr
Scalene Triangle √[s(s−a)(s−b)(s−c)], a+b+c

Where, s = (a+b+c)/2
Isosceles Triangle ½×b×h 2a + b
Section A Page 136

Isosceles Triangle ½×b×h 2a + b
Equilateral triangle (√3/4) × a2 3a
Right Angle Triangle ½ × b × h b + hypotenuse + h
Rhombus ½ × d1 × d2 4 × side
Parallelogram b×h 2(l+b)
Trapezium ½ h(a+c) a+b+c+d
Section A Page 137

Trapezium ½ h(a+c) a+b+c+d
Mensuration Formulas for 3D Shapes

Shape Volume (Cubic Curved Surface Area (CSA) or Lateral Surface Total Surface Area (TSA) Figure
units) Area (LSA) (Square units) (Square units)
Cube a3 LSA = 4 a2 6 a2
Cuboid l×b×h LSA = 2h(l + b) 2 (lb +bh +hl)
Sphere (4/3) π r3 4 π r2 4 π r2
Hemispher (⅔) π r3 2πr2 3πr2
Section A Page 138

Hemispher (⅔) π r3 2πr2 3πr2
e
Cylinder πr2h 2π r h 2πrh + 2πr2
Cone (⅓) π r2 h πrl πr (r + l)
Section A Page 139

Probability
Monday, 6 December, 2021 06:55 PM
Section A Page 140

Section A Page 141
Section A Page 142
Section A Page 143
Section A Page 144
Working with Proteins
wou.edu
Chapter 3: Investigating Proteins – Chemistry
124-157 minutes

3.6 References
Protein purification is a series of processes intended to isolate one or a few proteins from a complex mixture, usually cells, tissues or whole
organisms. Protein purification is vital for the characterization of the function, structure and interactions of the protein of interest. The purification
process may separate the protein and non-protein parts of the mixture, and finally separate the desired protein from all other proteins. Separation
of one protein from all others is typically the most laborious aspect of protein purification. Separation steps usually exploit differences in protein
size, physico-chemical properties, binding affinity and biological activity. The pure result may be termed protein isolate.
Protein purification is either preparative or analytical. Preparative purifications aim to produce a relatively large quantity of purified proteins for
subsequent use. Examples include the preparation of commercial products such as enzymes (e.g. lactase), nutritional proteins (e.g. soy protein
isolate), and certain biopharmaceuticals (e.g. insulin). Several preparative purifications steps are often deployed to removebi-products, such as
host cell proteins, which poses as a potential threat to the patient’s health. Analytical purification produces a relatively small amount of a protein
for a variety of research or analytical purposes, including identification, quantification, and studies of the protein’s structure, post-translational
modifications and function. Pepsin and urease were the first proteins purified to the point that they could be crystallized.
Extraction
If the protein of interest is not secreted by the organism into the surrounding solution, the first step of each purification process is the disruption of
the cells containing the protein. Depending on how fragile the protein is and how stable the cells are, one could, for instance, use one of the
following methods: i) repeated freezing and thawing, ii) sonication, iii) homogenization by high pressure (French press), iv) homogenization by
grinding (bead mill), and v) permeabilization by detergents (e.g. Triton X-100) and/or enzymes (e.g. lysozyme). Finally, the cell debris can be
removed by centrifugation so that the proteins and other soluble compounds remain in the supernatant.
Also proteases are released during cell lysis, which will start digesting the proteins in the solution. If the protein of interest is sensitive to
proteolysis, it is recommended to proceed quickly, and to keep the extract cooled, to slow down the digestion. Alternatively, one or more protease
inhibitors can be added to the lysis buffer immediately before cell disruption. Sometimes it is also necessary to add DNAse in order to reduce the
viscosity of the cell lysate caused by a high DNA content.
Precipitation and Differential Solubilization
In bulk protein purification, a common first step to isolate proteins is precipitation using a salt such as ammonium sulfate (NH4)2SO4. This process is
called Salting In or Salting Out (Figure 3.1) This is performed by adding increasing amounts of ammonium sulfate and collecting the different
fractions of precipitate protein. Ammonium sulfate is often used as it is highly soluble in water, has relative freedom from temperature effects and
typically is not harmful to most proteins. Furthermore, ammonium sulfate can be removed by dialysis (Figure 3.2). The hydrophobic groups on the
proteins get exposed to the atmosphere, attract other protein hydrophobic groups and get aggregated. Protein precipitated will be large enough to
be visible. One advantage of this method is that it can be performed inexpensively with very large volumes.
Working with Proteins Page 145

Figure 3.1 Salting In and Salting Out. During the salting in process, salt molecules increase the solubility of proteins by reducing the electrostatic
interactions between protein molecules. As the salt concentration is increased, protein-protein interactions become more energetically favorable
than protein-solvent interactions and the proteins precipitate from solution.
The first proteins to be purified are water-soluble proteins. Purification of integral membrane proteins requires disruption of the cell membrane in
order to isolate any one particular protein from others that are in the same membrane compartment. Sometimes a particular membrane fraction
can be isolated first, such as isolating mitochondria from cells before purifying a protein located in a mitochondrial membrane. A detergent such as
sodium dodecyl sulfate (SDS) can be used to dissolve cell membranes and keep membrane proteins in solution during purification; however,
because SDS causes denaturation, milder detergents such as Triton X-100 or CHAPS can be used to retain the protein’s native conformation during
complete purification.
Figure 3.2 Dialysis. The process of dialysis separates dissolved molecules by their size. The biological sample is placed inside a closed membrane,
where the protein of interest is too large to pass through the pores of the membrane, but through which smaller ions can easily pass. As the
solution comes to equilibrium, the ions become evenly distributed throughout the entire solution, while the protein remains concentrated in the
membrane. This reduces the overall salt concentration of the suspension.
Image adapted fromGjk003
Ultracentrifugation
Centrifugation is a process that uses centrifugal force to separate mixtures of particles of varying masses or densities suspended in a liquid. When a
vessel (typically a tube or bottle) containing a mixture of proteins or other particulate matter, such as bacterial cells, is rotated at high speeds, the
inertia of each particle yields a force in the direction of the particles velocity that is proportional to its mass. The tendency of a given particle to
move through the liquid because of this force is offset by the resistance the liquid exerts on the particle. The net effect of “spinning” the sample in a
centrifuge is that massive, small, and dense particles move outward faster than less massive particles or particles with more “drag” in the liquid.
When suspensions of particles are “spun” in a centrifuge, a “pellet” may form at the bottom of the vessel that is enriched for the most massive
particles with low drag in the liquid.
Non-compacted particles remain mostly in the liquid called “supernatant” and can be removed from the vessel thereby separating the supernatant
from the pellet. The rate of centrifugation is determined by the angular acceleration applied to the sample, typically measured in comparison to the
g. If samples are centrifuged long enough, the particles in the vessel will reach equilibrium wherein the particles accumulatespecifically at a point in
the vessel where their buoyant density is balanced with centrifugal force. Such an “equilibrium” centrifugation can allow extensive purification of a
given particle.
In sucrose gradient centrifugation, a linear concentration gradient of sugar (typically sucrose, glycerol, or a silica based density gradient media, like
Percoll) is generated in a tube such that the highest concentration is on the bottom and lowest on top. Percoll is a trademark owned by GE
Healthcare companies. A protein sample is then layered on top of the gradient and spun at high speeds in an ultracentrifuge. This causes heavy
macromolecules to migrate towards the bottom of the tube faster than lighter material. During centrifugation in the absence of sucrose, as
particles move farther and farther from the center of rotation, they experience more and more centrifugal force (the further they move, the faster
they move). The problem with this is that the useful separation range of within the vessel is restricted to a small observable window. A properly
designed sucrose gradient will counteract the increasing centrifugal force so the particles move in close proportion to the time they have been in
the centrifugal field. Samples separated by these gradients are referred to as “rate zonal” centrifugations. After separating the protein/particles,
the gradient is then fractionated and collected.

the gradient is then fractionated and collected.
Figure 3.3 Sucrose Density Gradient.

back to the top
Purification Strategy
Choice of a starting material is key to the design of a purification process. In a plant or animal, a particular protein usually isn’t distributed
homogeneously throughout the body; different organs or tissues have higher or lower concentrations of the protein. Use of only the tissues or
organs with the highest concentration decreases the volumes needed to produce a given amount of purified protein. If the protein is present in low
abundance, or if it has a high value, scientists may use recombinant DNA technology to develop cells that will produce large quantities of the
desired protein (this is known as an expression system). Recombinant expression allows the protein to be tagged, e.g. by a His-tag or Strep-tag to
facilitate purification, reducing the number of purification steps required. These techniques will be discussed in greater detail in Chapter 5.
An analytical purification generally utilizes three properties to separate proteins. First, proteins may be purified according to their isoelectric points
by running them through a pH graded gel or an ion exchange column. Second, proteins can be separated according to their size or molecular weight
via size exclusion chromatography or by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) analysis. Proteins are often purified
by using 2D-PAGE and are then analysed by peptide mass fingerprinting to establish the protein identity. This is very useful for scientific purposes
and the detection limits for protein are nowadays very low and nanogram amounts of protein are sufficient for their analysis. Thirdly, proteins may
be separated by polarity/hydrophobicity via high performance liquid chromatography or reversed-phase chromatography. Gel electrophoresis
techniques are discussed in more detail in Section 3.2. This section will focus predominantly on chromatographic separations.
For preparative protein purification, the purification protocol generally contains one or more chromatographic steps. The basic procedure in
chromatography is to flow the solution containing the protein through a column packed with various materials. Different proteins interact
differently with the column material, and can thus be separated by the time required to pass the column, or the conditions required to elute the
protein from the column. Usually proteins are detected as they are coming off the column by their absorbance at 280 nm. Many different
chromatographic methods exist, with the most common described below:
Size Exclusion Chromatography (also known as Gel Filtration Chromatography)
Chromatography can be used to separate protein in solution or under denaturing conditions by using porous gels. This technique is known as size
exclusion chromatography. The principle is that smaller molecules have to traverse a larger volume in a porous matrix. Consequentially, proteins of
a certain range in size will require a variable volume of eluent (solvent) before being collected at the other end of the column of gel. Thus, proteins
will be separated based on their size (Figure 3.4).
In the context of protein purification, the eluent is usually pooled in different test tubes. All test tubes containing no measurable trace of the
protein to purify are discarded. The remaining solution is thus made of the protein to purify and any other similarly-sized proteins.

protein to purify are discarded. The remaining solution is thus made of the protein to purify and any other similarly-sized proteins.
Figure 3.4 Size Exclusion Chromatography. Also known as Gel Filtration Chromatography, is a low resolution isolation method that involves the use
of beads that have tiny “tunnels” in them that each have a precise size. The size is referred to as an “exclusion limit,” which means that molecules
above a certain molecular weight will not fit into the tunnels. Molecules with sizes larger than the exclusion limit do not enter the tunnels and pass
through the column relatively quickly by making their way between the beads. Smaller molecules, which can enter the tunnels, do so, and thus,
have a longer path that they take in passing through the column. Because of this, molecules larger than the exclusion limit will leave the column
earlier, while smaller molecules that pass through the beads will elute from the column later. This method allows separation of molecules by their
size.
Image fromDr. Kevin Ahern and Indira Rajagopal
Hydrophobic Interaction Chromatography (HIC)

HIC media is amphiphilic, with both hydrophobic and hydrophilic regions, allowing for separation of proteins based on their surface hydrophobicity.
Target proteins and their product aggregate species tend to have different hydrophobic properties and removing them via HIC further purifies the
protein of interest. Additionally, the environment used typically employs less harsh denaturing conditions than other chromatography techniques,
thus helping to preserve the protein of interest in its native and functional state. In pure water, the interactions between the resin and the
hydrophobic regions of protein would be very weak, but this interaction is enhanced by applying a protein sample to HIC resin in high ionic strength
buffer. The ionic strength of the buffer is then reduced to elute proteins in order of decreasing hydrophobicity (Figure 3.5).
Figure 3.5 Hydrophobic Interaction Chromatography. The column matrix, shown in blue has a hydrophobic ligand covalently attached. In high salt
conditions, proteins will bind to the matrix with differing affinity, with more hydrophobic proteins (shown in yellow) binding more tightly than more
hydrophilic proteins (shown in green) When the salt concentration is decreased, proteins that are more hydrophilic will be released first, followed
more hydrophobic proteins.
Ion Exchange Chromatography

Ion exchange chromatography separates compounds according to the nature and degree of their ionic charge. The column to be used is selected
according to its type and strength of charge. Anion exchange resins have a positive charge and are used to retain and separate negatively charged
compounds (anions), while cation exchange resins have a negative charge and are used to separate positively charged molecules (cations).
Before the separation begins a buffer is pumped through the column to equilibrate the opposing charged ions. Upon injection of the sample, solute

Before the separation begins a buffer is pumped through the column to equilibrate the opposing charged ions. Upon injection of the sample, solute
molecules will exchange with the buffer ions as each competes for the binding sites on the resin. The length of retention for each solute depends
upon the strength of its charge. The most weakly charged compounds will elute first, followed by those with successively stronger charges. Because
of the nature of the separating mechanism, pH, buffer type, buffer concentration, and temperature all play important roles in controlling the
separation.
Figure 3.6 demonstrates a type of ion exchange column known as a cation exchange column. In this case, the support consists of tiny beads to
which are attached chemicals possessing a charge. Each charged molecule has a counter-ion. The figure shows the beads (blue) with negatively
charged groups (red) attached. In this example, the counter-ion is sodium, which is positively charged. The negatively charged groups are unable to
leave the beads, due to their covalent attachment, but the counter- ions can be “exchanged” for molecules of the same charge. Thus, a cation
exchange column will have positively charged counter-ions and positively charged compounds present in a mixture passed through the column will
exchange with the counter-ions and “stick” to the negatively charged groups on the beads. Molecules in the sample that are neutral or negatively
charged will pass quickly through the column. On the other hand, in anion exchange chromatography, the chemical groups attached to the beads
are positively charged and the counter-ions are negatively charged. Molecules in the sample that are negatively charged will “stick” and other
molecules will pass through quickly. To remove the molecules “stuck” to a column, one simply needs to add a high concentration of the appropriate
counter-ions to displace and release them. This method allows the recovery of all components of the mixture that share the same charge.
Ion exchange chromatography is a very powerful tool for use in protein purification and is frequently used in both analytical and preparative
separations.
Figure 3.6 Cation Exchange Chromatography. In this diagram the negatively charged molecules (shown in red) are covalently attached to the
column matrix beads (shown in blue). Sodium ions (Na+) are the counter ions that are replaced by positively charged proteins within the protein
mixture. Neutral and negatively charged proteins do not stick and will pass through the column. The positively charged proteins can then be eluted
from the column by adding higher concentrations of the counter ion (in this case the sodium ions).
Image fromKevin Ahern and Indira Rajagopal
Affinity Chromatography is a separation technique based upon molecular conformation, which frequently utilizes application specific resins. These
resins have ligands (small molecules) attached to their surfaces which are specific for and will bind with the compounds to be separated. Most
frequently, these ligands function in a fashion similar to that of antibody-antigen interactions. This “lock and key” fit between the ligand and its
target compound makes it highly specific, frequently generating a single peak, while all else in the sample is unretained (Figure 3.7).
For example, many membrane proteins are glycoproteins and can be purified by lectin affinity chromatography. Detergent-solubilized proteins can
be allowed to bind to a chromatography resin that has been modified to have a covalently attached lectin. Proteins that do not bind to the lectin
are washed away and then specifically bound glycoproteins can be eluted by adding a high concentration of a sugar that competes with the bound
glycoproteins at the lectin binding site. Some lectins have high affinity binding to oligosaccharides of glycoproteins that is hard to compete with
sugars, and bound glycoproteins need to be released by denaturing the lectin.

sugars, and bound glycoproteins need to be released by denaturing the lectin.
Figure 3.7 Example of Affinity Chromatography. In this example, protein P1 has affinity for ligand Z and will bind to the column while proteins P2
and P3 will pass through the column. Protein P1 can then be eluted from the column using high concentrations of free ligand Z.
A common technique involves engineering a sequence of 6 to 8 histidine residues into the N- or C-terminal of a recombinant protein. The
polyhistidine binds strongly to divalent metal ions such as nickel and cobalt. The protein can be passed through a column containing immobilized
nickel ions, which binds the polyhistidine tag. All untagged proteins pass through the column. The protein can be eluted with imidazole, which
competes with the polyhistidine tag for binding to the column, or by a decrease in pH (typically to 4.5), which decreases the affinity of the tag for
the resin. While this procedure is generally used for the purification of recombinant proteins with an engineered affinity tag (such as a 6xHis tag), it
can also be used for natural proteins with an inherent affinity for divalent cations.
Immunoaffinity chromatography
A special type of affinity chromatography is Immunoaffinity chromatography (Figure 3.8). This technique uses the specific binding of an antibody
with its antigen (target molecule that the antibody will bind with selectively) to purify the protein of interest. The procedure involves immobilizing
an antibody to a solid substrate (e.g. a porous bead or a membrane), which then selectively binds the target, while everything else flows through.
The target protein can be eluted by changing the pH or the salinity. The immobilized ligand can be an antibody (such as Immunoglobulin G) or it can
be a protein (such as Protein A). Because this method does not involve engineering in a tag, it can be used for proteins from natural sources.
Antibody structure and their use in protein identification will be discussed in greater detail in Section 3.2.

Antibody structure and their use in protein identification will be discussed in greater detail in Section 3.2.
Figure 3.8. An Antigen Immunoprecipitation Experiment. The antibody is either pre-immobilized to a solid support (left) or immobilized using
antibody binding proteins after incubation with the sample (right). Immobilization allows the immune complex to be extracted from the complex
sample, washed and eluted providing a high enrichment of the protein under investigation
back to the top
High Performance Liquid Chromatography (HPLC) and Fast Protein Liquid Chromatography (FPLC)
High performance liquid chromatography or high pressure liquid chromatography (HPLC) is a form of chromatography applying high pressure to
drive the solutes through the column faster. This means that the diffusion is limited and the resolution is improved. The most common form is
“reversed phase” HPLC, where the column material is hydrophobic. The proteins are eluted by a gradient of water and increasing amounts of an
organic solvent, such as acetonitrile. The proteins elute according to their hydrophobicity. After purification by HPLC the protein is in a solution that
only contains volatile compounds, and can easily be lyophilized (freeze dried). HPLC purification frequently results in denaturation of the purified
proteins and is thus not applicable to proteins that do not spontaneously refold.
Due to the drawbacks of HPLC, an alternative technique using a lower pressure system was developed and is called Fast protein liquid
chromatography (FPLC). FPLC is a form of liquid chromatography that is often used to analyze or purify mixtures of proteins. As in other forms of
chromatography, separation is possible because the different components of a mixture have different affinities for two materials, a moving fluid
(the “mobile phase”) and a porous solid (the stationary phase). In FPLC the mobile phase is an aqueous solution, or “buffer”. The buffer flow rate is
controlled by a positive-displacement pump and is normally kept constant, while the composition of the buffer can be varied by drawing fluids in
different proportions from two or more external reservoirs. The stationary phase is a resin composed of beads, usually of cross-linked agarose,
packed into a cylindrical glass or plastic column. FPLC resins are available in a wide range of bead sizes and surface ligands depending on the
application.
In the most common FPLC strategy, an ion exchange resin is typically chosen (Figure 3.9). A mixture containing one or more proteins of interest is
dissolved in 100% buffer A and pumped into the column. The proteins of interest bind to the resin while other components are carried out in the
buffer. The total flow rate of the buffer is kept constant; however, the proportion of Buffer B (the “elution” buffer) is gradually increased from 0%
to 100% according to a programmed change in concentration (the “gradient”). Buffer B contains high concentrations of the exchanger ion. Thus as
the concentration of the Buffer B gradually increases, bound proteins will dissociate depending on their ionic interactions with the column matrix
and appear in the eluant. The eluant passes through two detectors which measure salt concentration (by conductivity) and protein concentration
(by absorption of ultraviolet light at a wavelength of 280nm). As each protein is eluted it appears in the eluant as a “peak” in protein concentration
and can be collected for further use.
FPLC was developed and marketed in Sweden by Pharmacia in 1982 and was originally called fast performance liquid chromatography to contrast
it with HPLC or high-performance liquid chromatography. FPLC is generally applied only to proteins; however, because of the wide choice of resins
and buffers it has broad applications. In contrast to HPLC the buffer pressure used is relatively low, typically less than 5 bar, but the flow rate is
relatively high, typically 1-5 ml/min. FPLC can be readily scaled from analysis of milligrams of mixtures in columns with a total volume of 5 ml or less

relatively high, typically 1-5 ml/min. FPLC can be readily scaled from analysis of milligrams of mixtures in columns with a total volume of 5 ml or less
to industrial production of kilograms of purified protein in columns with volumes of many liters.
Figure 3.9 Typical FPLC System. A. Scheme of basic compounents and typical flow path for a chromatography system. B. Picrue of GE Healthcare
AKTA FPLC apparatus.
Image provided byLaVerde, V., Dominici, P. and Astegno, A. (2017) Bio-protocol 7(8): e2230.
Purification Scheme
During the protein purification process it is necessary to have a quantitative system to determine how much protein has been purified, what
concentration the protein represents from the original mixture, how biologically active the purified protein is, and the overall purity of the protein.
This will help guide and optimize the purification method being developed. Ineffective separation techniques can be disregarded and other
techniques that give higher yield or that retain biologically activity of the protein can be adopted.
Thus, each step in the purification scheme is quantitatively evaluated for the following parameters: total protein, total activity, specific activity,
yield, purification level. Each of these parameters will be defined within the sample protocol given below.
Pretend you are a researcher that wants to isolate a novel, unknown protein from a bacterial culture. You grow 500 ml of the bacteria overnight at
37oC and harvest the bacteria by centrifugation. You remove the culture broth and retain the bacterial pellet. You then lyse the bacteria using
freeze/thaw in 10 mL of reaction buffer. You then centrifuge the lysed bacteria to remove the insoluble materials and retain the supernatent that
contains the soluble proteins. Your protein of interest has a biological activity that you can measure using a simple assay that causes a color change
in the reaction mixture (Figure 3.10). You also note that this reaction rate increases with increasing concentrations of your protein supernatent
(Figure 3.10)
Figure 3.10. Example of a Chemical Reaction that causes a color change from orange to brown depending on increasing concentration.
Image from: Ludwig, N., et. al. (2015) on Research Gate
At this point, you can measure your baseline concentrations for the first purification level (bacterial lysis and removal of insoluble proteins and
other cellular debris by centrifugation).
Total Protein is calculated by measuring the concentration in a fraction of your sample, and then multiplying that by the total volume of your
sample. In this case, you are starting with 10 mL of supernatent. In a typical assay to measure protein concentration, you will use 50 – 200 μL of
sample to determine the protein concentration. For example, if you calculate that there is 7.5 μg/μL in your initial assay, you would need to convert
that value into mg/mL and then multiply it by 10 mL for a total of 75 mg of protein in 10 mL of supernatant (Table 3.1)
Total Activity is measured as the enzyme activity within the assay, multiplied by the total volume of the sample. For example, in the initial sample,
you might use 5 to 50 μL of sample in your biological reaction (Figure 3.10). If you calculated the activity in your assay to be 2.5 units/μL, this would
be equivalent to 2,500 units/mL or 25,000 units/10 mL of supernatant. Note that, the enzyme unit, or international unit for enzyme (symbol U,
sometimes also IU) is a unit of enzyme’s catalytic activity. 1 U (μmol/min) is defined as the amount of the enzyme that catalyzes the conversion of
one micromole of substrate per minute under the specified conditions of the assay method.
Specific Activity is measured by dividing the Total Activity by the Total Protein. In our example, 25,000 units divided by 75 mg of protein = 333.3
units/mg.
Yield is a measure of the biological activity retained in the sample after each purification step. The amount in the first step is set to be 100%. All
subsequent yield steps will be evaluated using the first purification step. It is calculated by dividing the total activity of the current step, by the total
activity of the first step and then multiplying by 100.
Purification level evaluates the purity of the protein of interest by dividing the specific activity calculated after each purification step by the specific
activity of the first purification step. Thus, the first step always has a value of 1.
Table 3.1 Typical Protein Purification Scheme

Note that after each purification step that the Total Protein goes down, as you are purifying your protein away from other proteins in the mixture.
Total Activity also goes down with each purification step, as some of your protein of interest is also lost at each purification step, because (1) some
protein will stick to the test tubes and glassware, (2) some protein won’t bind with 100% efficiency to your column matrix, (3) some protein may
bind too tightly to be removed from the column matrix during elution, and (4) some protein may be denatured or degraded during the purification
process.
The amount of your protein of interest that is lost is represented within the overall percent yield for each purification step. If the percent yield is
too low alternative purification methods should be explored.
Note that in a good protein purification scheme that the specific activity should go up substantially with each level of purification as the amount of
your protein of interest makes up a greater percentage of the total protein within that fraction. If the specific activity only increases modestly
within a purification step, or if it decreases during a purification step, this could indicate that (1) your protein of interest is being substantially lost at
that step, (2) that your protein of interest is being denatured or degraded and is no longer biologically active, or (3) that a required cofactor or
binding protein is being reduced at that purification step. Additional experiments may need to be conducted to determine which of the causes
predominates, so that steps can be taken to reduce protein inactivation. For example, many proteins are temperature sensitive and will degrade or
denature at room temperature. Completing purification steps on ice can often reduce degradation.
Overall, the fold increase in purification level should increase exponentially during the purification process. Note that in our example, if after 4
steps of purification our proteins is close to 95% pure, this would indicate that our protein of interest makes up approximately 1.24% of the total
protein within the sample.
back to the top

Analytical techniques that can be used to positively identify or visualize a protein of interest within a mixture can also be a valuable tool to
understanding the biological activity and significance of a protein within a living system and can also be used to help guide protein purification
schemes.
Gel Electrophoresis
(work derived fromMagdeldin, S.)
Agarose is a natural linear polymer extracted from seaweed that forms a gel matrix by hydrogen-bonding when heated in a buffer and allowed to
cool. For most applications, only a single-component agarose is needed and no polymerization catalysts are required (Figure 3.11). Therefore,
agarose gels are simple and rapid to prepare. They are the most popular medium for the separation of moderate and large-sized nucleic acids and
have a wide range of separation but a relatively low resolving power, since the bands formed in the gels tend to be fuzzy and spread apart. This is a
result of pore size and cannot be largely controlled. These and other advantages and disadvantages of using agarose gels for electrophoresis are
summarized in Table 3.2. Agarose gels are not typically used for protein samples and won’t be discussed in this chapter further. However, they will
be revisted in Chapter 5 covering nucleic acid techniques.
Table 3.2. Advantages and Disadvantages of Agarose Gel Electrophoresis.
Polyacrylamide gels are chemically cross-linked gels formed by the polymerization of acrylamide with a cross-linking agent, usually N,N’-
methylenebisacrylamide (Figure 3.11). The reaction is a free radical polymerization, usually carried out with ammonium persulfate as the initiator
and N,N,N’,N’-tetramethylethylendiamine (TEMED) as the catalyst. Although the gels are generally more difficult to prepare and handle, involving a
longer time for preparation than agarose gels, they have major advantages over agarose gels. They have a greater resolving power, can
accommodate larger quantities of sample without significant loss in resolution and the purity of the sample recovered from polyacrylamide gels is
extremely high. Moreover, the pore size of the polyacrylamide gels can be altered in an easy and controllable fashion by changing the
concentrations of the two monomers. Thus, it is commonly used to separate proteins and smaller fragments of DNA. It should be noted that
polyacrylamide is a neurotoxin (when unpolymerized), but with proper laboratory care it is no more dangerous than various commonly used
chemicals. Some advantages and disadvantages of using polyacrylamide gels for electrophoresis are depicted in Table 3.3.
Table 3.3. Advantages and Disadvantages of Polyacrylamide Gel Electrophoresis.

Hydrated gel networks have many desirable properties for electrophoresis. They allow a wide variety of mechanically stable experimental formats
such as horizontal/vertical electrophoresis in slab gels or electrophoresis in tubes or capillaries. The mechanical stability also facilitates post
electrophoretic manipulation making further experimentation possible such as blotting, electro-elution or mass spectral identification /finger
printing of intact proteins or of proteins digested in gel slices. Since gels used in biochemistry are chemically rather unreactive, they interact
minimally with biomolecules during electrophoresis allowing separation based on physical rather than chemical differences between sample
components.
Figure 3.11 Gels Commonly Used in Electrophoresis. (A) Agarose is composed of agarbiose, (B) The polymerization of acrylamide and bisacrylamide
to form polyacrylamide gel. The polymerization reaction is initiated by persulfate radicals and catalyzed by TEMED.
Gel electrophoresis of proteins with a polyacrylamide matrix, commonly called polyacrylamide gel electrophoresis (PAGE) is undoubtedly one of
the most widely used techniques to characterize complex protein mixtures. It is a convenient, fast and inexpensive method because they require
only the order of micrograms quantities of protein. They are usually run in a vertical format and the gel rigs contain an upper and lower buffer
reservoir (Fig. 3.12A). The samples are loaded in wells that contact the upper buffer reservoir which will house the negative cathode. The proteins
migrate towards the positive anode when the electric current is applied.
Note that proteins have a net electrical charge if they are in a medium having a pH different from their isoelectric point and therefore have the
ability to move when subjected to an electric field. The migration velocity is proportional to the ratio between the charges of the protein and its
mass. The higher charge per unit of mass the faster the migration.
Proteins do not have a predictable structure as nucleic acids, and thus their rates of migration are not similar to each other. Furthermore, they will
not migrate when applying an electromotive force, when the pH of the system is the same as isoelectric point. PAGE gels that are run in this fashion
are called Native PAGE, as the proteins are still folded in their native state found in vivo. In this situation, proteins migrate according to their
charge, size and shape.
Alternatively, proteins may be denatured prior to electrophoresis. The most common way to denature the proteins is by adding a detergent such as
sodium dodecyl sulfate (SDS) (Fig 3.12B). This not only denatures the proteins, but it also coats the protein with a negative charge, such that all of
the proteins will run towards the positive lead when placed into an electric field. This type of electrophoresis is referred to as SDS-PAGE and
separates proteins exclusively according to molecular weight. A reducing agent that breaks disulfide bonds, such as dithiothreitol (DTT) is often
added to the loading buffer as well, causing proteins to fully denature and dissociate into the monomer subunits (Fig 3.12C). This ensures that the
proteins migrate through the gel in direct relation to their size, rather than by charge or shape.

Figure 3.12 Polyacrylamide Gel Electrophoresis (PAGE). (A) Shows a typical set up for PAGE. A vertical gel is placed into a rig with an upper and
lower buffer reservoir. The upper reservoir contains the gel wells where the protein is loaded and will house the cathode (negative charge). The
proteins will run towards the anode (positive charge) when an electric field is placed on the system, in relation to the protein size, shape and
charge. (B) Sodium dodecyl sulfate (SDS) is often used to denature proteins prior to PAGE analysis, causing proteins to migrate based on size only.
(C) Reducing agents, such as dithiothreitol (DTT) are often used in combination with SDS, to ensure that disulfide bonds within the protein or
between protein subunits are fully reduced to free cysteine residues.
Figures from: (A) Bensaccount (B) Fdardel,and (C) Edgar181
Detection of Proteins in Gels

Proteins separated on a polyacrylamide gel can be detected by various methods, for instance dyes and silver staining (Figure 3.13).
• Dyes
The Coomassie blue staining allows detecting up to 0.2 to 0.6 µg of protein, and is quantitative (linear) up to 15 to 20 µg. It is often used in
methanol-acetic acid solutions and is discolored in isopropanol-acetic acid solutions (Fig. 1 A). For staining of 2-DE gels it is recommended to
remove ampholytes by adding trichloroacetic (TCA) to the dye and subsequently discolor with acetic acid.
• Silver staining
It is an alternative to routine staining protein gels (as well as nucleic acids and lipopolysaccharides) because its ease use and high sensitivity (50 to
100 times more sensitive than Coomassie blue staining) (Fig. 1 B). This staining technique is particularly suitable for two-dimensional gels.
• Detection of radioactive proteins by autoradiography
The autoradiography is a detection technique of radioactively labeled molecules that uses photographic emulsions sensitive to radioactive particles
or light produced by an intermediate molecule. The emulsion containing silver is sensitive to particulate radiation (alpha, beta) or electromagnetic
radiation (gamma, light…), so that it precipitates as metallic silver. The emulsion will develop as dark precipitates in the region in which radioactive
proteins are detected.

proteins are detected.
Figure 3.13. SDS-PAGE. Proteins separated on SDS-PAGE and detected by Coomassie blue (A) and silver staining (B). Standards of proteins to know
molecular weight are also loaded at edges.
Isoelectric Focusing
This technique is based on the movement of molecules in a pH gradient. Amphoteric molecules such as amino acids and proteins are separated in
an environment where there is a difference of potential and pH gradient. The region of the anode (+) is acidic and the cathode (-) is alkaline.
Between them down a pH gradient such that the molecules to be separated have their isoelectric point within the range. Substances that are
initially in regions with a pH below its isoelectric point are positively charged and migrate towards the cathode, while those that are in media with
pH lower than its pI will have negative charge and migrate towards the anode (Figure 3.14). The migration will lead to a region where the pH
coincide with its pI, have a zero net charge (form zwitterions) and stop. Thus amphoteric molecules are located in narrow bands where the pI
coincides with the pH. In this technique the point of application is not critical as molecules will always move to their pI region. The stable pH
gradient between the electrodes is achieved using a mixture of low molecular weight ampholytes which pI covers a preset range of pH.
Figure 3.14. Isoelectric Focusing. A pH gradient is established in a gel before loading the sample. After the sample is loaded a voltage is applied. The
protein will migrate to their isoelectric pH, which they have no net charge.
Two-Dimensional Gel Electrophoresis

Two-dimensional gel electrophoresis (2-DE) is based on separating a mixture of proteins according to two molecular properties, one in each
dimension. The most used is based on a first dimension separation by isoelectric focusing and second dimension according to molecular weight by
SDS-PAGE (Figure 3.15).
The general workflow in a 2-DE experiment would be:
• Sample Preparation
The method of sample preparation depends on the aim of the research and is crucial to the success of the experiment. Factors such as the
solubility, size, charge, and isoelectric point (pI) of the proteins of interest enter into sample preparation. Sample preparation is also important in
reducing the complexity of a protein mixture. The protein fraction to be loaded on a 2-DE gel must be in a low ionic strength denaturing buffer that
maintains the native charges of proteins and keeps them soluble.
• First-Dimension Separation
This part is performed by IEF. Using this technique, proteins are separated on the basis of their pI, the pH at which a protein carries no net charge
and will not migrate in an electrical field.
• Equilibration
A conditioning step is applied to proteins separated by IEF prior to the second-dimension run. This process reduces disulfide bonds and alkylates the
resultant sulfhydryl groups of the cysteine residues. Concurrently, proteins are coated with SDS for separation on the basis of molecular weight.
• Second-Dimension Separation
This part is performed by SDS-PAGE. The choice for the gel depends on the protein molecular weight range to be separated. The ability to run many

This part is performed by SDS-PAGE. The choice for the gel depends on the protein molecular weight range to be separated. The ability to run many
gels at the same time and under the same conditions is important for the purpose of gel-to-gel comparison.
• Staining
In order to visualize proteins in gels, they must be stained in some manner. The selection of staining method is determined by several factors,
including desired sensitivity, linear range, ease of use, expense, and the type of imaging equipment available. At present there is no ideal universal
stain. Sometimes proteins are detected after transference to a membrane support by western blotting, which is described in more detail below.
• Image Analysis
The ability to collect data in digital form is one of the major factors that enable 2-DE gels to be a practical means of collecting proteome
information. It allows unprejudiced comparison of gels and cataloging of immense amounts of data. Many types of imaging devices interface with
software designed specifically to collect, interpret, and compare proteomics data. One of the biggest problems in 2-DE is the analysis and
comparison of complex mixtures of proteins. Currently there are databases capable of comparing two-dimensional gel patterns. These systems
allow automatic comparison of spots for the precise identification of those needed in the quantitative analysis.
• Protein Identification
Once interesting proteins are selected by differential analysis or other criteria, the proteins can be excised from gels, distained and digested to
prepare their identification by mass spectrometry. This technique is known as peptide mass fingerprinting. The ability to precisely determine
molecular weight by matrix-assisted laser desorption/ionization- time of flight mass spectrometry (MALDI-TOF MS) and to search databases for
peptide mass matches has made high-throughput protein identification possible. Proteins not identified by MALDI- TOF can be identified by
sequence tagging or de novo sequencing using the Q-TOF electrospray LC-MS-MS.
Fig. 3.15 Two-Dimentional Gel Electrophoresis. Proteins of Chlamydomonas reinhardtii resolved by 2-DE from preparative gels stained with MALDI-
MS compatible silver reagent for peptide mass fingerprinting analysis. First dimension: isoelectric focusing in a 3-11 pH gradient. Second dimension:
SDS-PAGE in a 12% acrylamide (2.6% crosslinking) gel (1.0 mm thick). Numbered spots marked with circle correspond to proteins compared to be
subsequently identified by MALDI-TOF MS. The MALDI-TOF MS analysis of protein sequences is discussed in more detail in section 3.3 below.
back to the top
Antibody Structure and Production

(work derived fromCharles Molnar and Jane Gair)
An antibody, also known as an immunoglobulin (Ig), is a protein that is produced by plasma cells after stimulation by an antigen. Antibodies are the
functional basis of humoral immunity. Antibodies occur in the blood, in gastric and mucus secretions, and in breast milk. Antibodies in these bodily
fluids can bind pathogens and mark them for destruction by phagocytes before they can infect cells. The molecule that is bound by an antibody is
termed the antigen. Antibodies are highly specific for a single antigen or a group of antigens that share highly conserved structural features.
Proteins can act as antigens that are recognized by antibodies. Thus, within the field of biochemistry and molecular biology, antibodies are used as
important tools that help us to determine the function and expression pattern of proteins. They can also be used therapeutically in the treatment
of diseases such as cancer.
Antibody Structure

Antibody Structure
The most common type of antibody used in biochemical methodologies is known as immunoglobulin G (IgG) and will be the focus of this section. An
IgG antibody molecule is comprised of four polypeptides: two identical heavy chains (large peptide units) that are partially bound to each other in a
“Y” formation, which are flanked by two identical light chains (small peptide units), as illustrated in Figure 3.16. Bonds between the cysteine amino
acids in the antibody molecule attach the polypeptides to each other. The areas where the antigen is recognized on the antibody are variable
domains and the antibody base is composed of constant domains.
Figure 3.16 Immunoglobulin G (IgG) Structure(a) As a germ-line B cell matures, an enzyme called DNA recombinase randomly excises V and J
segments from the light chain gene. Splicing at the mRNA level results in further gene rearrangement. As a result, (b) each mature B cell produces a
single antibody that has a unique variable region capable of binding a different antigen.
Image from: Charles Molnar and Jane Gair
In germ-line B cells, the variable region of the light chain gene has 40 variable (V) and five joining (J) segments. An enzyme called DNA recombinase
randomly excises most of these segments out of the gene during B cell maturation, and splices one V segment to one J segment. During RNA
processing, all but one V and J segment are spliced out. Recombination and splicing may result in over 106 possible VJ combinations! As a result,
each differentiated B cell in the human body typically has a unique variable chain that will recognize a unique antigen. The constant domain, which
does not bind antibody, is the same for all antibodies.
Production of Polyclonal Antibodies
Antibodies used for research and diagnostic purposes are often obtained by injecting a lab animal such as a rabbit or a goat with a specific antigen.
Within a few weeks, the animal’s immune system will produce high levels of antibodies specific for the antigen. These antibodies can be harvested
in an antiserum, which is whole serum collected from an animal following exposure to an antigen. Because most antigens are complex structures
with multiple epitopes, they result in the production of multiple antibodies in the lab animal. This so-called polyclonal antibody response is also
typical of the response to infection by the human immune system. Antiserum drawn from an animal will thus contain antibodies from multiple
clones of B cells, with each B cell responding to a specific epitope on the antigen (Figure 3.17).

clones of B cells, with each B cell responding to a specific epitope on the antigen (Figure 3.17).
Figure 3.17. Polyclonal Antibody Production. This diagram illustrates the process for harvesting polyclonal antibodies produced in response to an
antigen.
Image from Polyclonal and Monoclonal Antibody Production
Lab animals are usually injected at least twice with antigen when being used to produce antiserum. The second injection will activate memory cells
that make class IgG antibodies against the antigen. The memory cells also undergo affinity maturation, resulting in a pool of antibodies with higher
average affinity. Affinity maturation occurs because of mutations in the immunoglobulin gene variable regions, resulting in B cells with slightly
altered antigen-binding sites. On re-exposure to the antigen, those B cells capable of producing antibody with higher affinity antigen-binding sites
will be stimulated to proliferate and produce more antibody than their lower-affinity peers. An adjuvant, which is a chemical that provokes a
generalized activation of the immune system that stimulates greater antibody production, is often mixed with the antigen prior to injection.
Antiserum obtained from animals will not only contain antibodies against the antigen artificially introduced in the laboratory, but it will also contain
antibodies to any other antigens to which the animal has been exposed during its lifetime. For this reason, antisera must first be “purified” to
remove other antibodies before using the antibodies for research or diagnostic assays.
Production of Monoclonal Antibodies
Some types of assays require better antibody specificity and affinity than can be obtained using a polyclonal antiserum. To attain this high
specificity, all of the antibodies must bind with high affinity to a single epitope. This high specificity can be provided by monoclonal antibodies
(mAbs). Table 3.4 compares some of the important characteristics of monoclonal and polyclonal antibodies.
Table 3.4 Comparison of Monoclonal and Polyclonal Antibodies
Unlike polyclonal antibodies, which are produced in live animals, monoclonal antibodies are produced in vitro using tissue-culture techniques. mAbs
are produced by immunizing an animal, often a mouse, multiple times with a specific antigen. B cells from the spleen of the immunized animal are
then removed. Since normal B cells are unable to proliferate forever, they are fused with immortal, cancerous B cells called myeloma cells, to yield
hybridoma cells. All of the cells are then placed in a selective medium that allows only the hybridomas to grow; unfused myeloma cells cannot
grow, and any unfused B cells die off. The hybridomas, which are capable of growing continuously in culture while producing antibodies, are then
screened for the desired mAb. Those producing the desired mAb are grown in tissue culture; the culture medium is harvested periodically and
mAbs are purified from the medium. This is a very expensive and time-consuming process. It may take weeks of culturing and many liters of media
to provide enough mAbs for an experiment or to treat a single patient. mAbs are expensive (Figure 3.18).

to provide enough mAbs for an experiment or to treat a single patient. mAbs are expensive (Figure 3.18).
Figure 3.18. Monoclonal Antibodies (mAbs) are produced by introducing an antigen to a mouse and then fusing polyclonal B cells from the mouse’s
spleen to myeloma cells. The resulting hybridoma cells are cultured and continue to produce antibodies to the antigen. Hybridomas producing the
desired mAb are then grown in large numbers on a selective medium that is periodically harvested to obtain the desired mAbs.
Image fromPolyclonal and Monoclonal Antibody Production
back to the top
Enzyme-Linked Immunosorbent Assay (ELISA) and Microarrays

Since the very first use of antibodies for the detection of antigens, many different technologies have been developed that make use of the
antibodies’ capability to bind to other molecules. During the 1950s, the scientists Yalow and Berson developed a method where radioactivity is used
to determine the amount of an analyte in a solution. This so called ‘radioimmunoassay’ (RIA), for which Yarlow received the Nobel prize in 1977,
was a very sensitive method for the detection of hormones but using radioactivity for antigen detection is not safe and suitable for a general use.
Hence, an alternative procedure was developed by linking enzymes to antibodies instead of a radioactive molecule, and by adhering molecules to
surfaces. In one of the nowadays most common applications today are measuring the quantity of a biomolecule in a sample by “enzyme-linked
immunosorbent assay” (ELISA). This term originally refers to the use of an enzyme to report an interaction between an antibody and its binding
partner (Gan & Patel, 2013). The foundation for Perlmann and Engvall in Sweden (Engvall & Perlmann, 1971)as well as Schuurs and van Weemen
from the Netherlands (Van Weemen & Schuurs, 1971), who built assays with immobilized and enzyme-modified reagents in the early 1970s. Today,
scientists also use colored molecules (so called fluorophores) that re-emit light upon excitation to visualize antibody-antigen interactions. Many
variants of experimental procedures have been developed, and it is common to build assays using more than one antibody to detect a target of
interest (see Figure 3.xx C-D). To further enhance the possibilities offered by the immunoassay format, applications based on microarrays have
been developed and which allow to measure more than one molecule in a single reaction chamber (see below).
ELISA Assay Design
The use of antibodies allows designing experiments in many different ways for the intended analysis. To achieve the best possible results from the
experiment, different reagents, additives, and solutions have to be tested for their optimal combination and concentration, incubation times and
the number of wash cycles need to be evaluated and adjusted. This is to avoid unwanted interactions, which disturb the analysis from detecting the
target of interest. Moreover, the mode of how a target is identified and detection can be performed in a number of ways, as described in Figure
3.19

3.19
Figure 3.19. Different setups for ELISA and Other Immunossays. In ELISA assays, the antibodies may (A) detect an immobilized antigen, (B) capture
a labeled antigen, (C) capture an unlabeled antigen and use a second, labeled antibody to detect the captured antigen, or (D) use a third antibody
for detection, or even use two antibodies for detection (E). Direct labeling of the antibody or antigen as in (A), (B), and (C) is the simplest and fastest
method for detection. Using a secondary antibody as detection method, as shown in (D) and (E), will further increase the sensitivity and selectivity
of the analysis. The method used in (D) also allows greater flexibility, whereas method (E) further increases the specificity, as three antibodies must
bind the antigen in order to produce a reporter molecule. Out of the presented assays, the most commonly used concepts are shown in (C) and (D).
Multiplexing
A new era in immunoassays started with the development of a technology called microarrays. The term microarray most commonly describes the
ordered organization of small volume droplets that have dried on a small surface area. The reaction dimensions are miniaturized so that many
assays can be performed in multiple samples in parallel, several thousands of different features may be presented to the surrounding solution. This
means that scientists can measure a large number of molecules with one single experiment. There is the possibility to use microscope glass slides
and specialized robotics that deposit very small drops of liquid (1 nl = 0.000000001 liter) on the glass surface in an ordered fashion. This leaves
behind spots of less than one millimeter in diameter (0.15 mm). Another common technique for multiplexing is to use even smaller and color-coded
particles (diameter of 0.005 mm). These particles can be coated with antibodies to fish out the analyte from the solution.
Sensitivity
In many applications it is important to measure very small amounts (sometimes only traces) of a molecule in a given sample. In order to achieve the
required sensitivity, the conditions of the experiment need to be adjusted to suit the antibodies, the detection system, and the type of samples. In
addition, there is progress being made on using better colors,signal amplification specialized lasers and filters, as well asminiaturization(Ekins &
Edwards, 1997).
Specific examples
There are many examples of how ELISA assays may be used in basic research and in clinical diagnostics. One specific example is the sensitive
sandwich-type enzyme-linked immunoassay used to determine the amount of the protein prostate-specific antigen (PSA), which is a biomarker
used to detect prostate cancer(Kuriyama et al., 1980).
Microarray assays on the other hand, have previously received a lot of attention for their use in parallel analysis of DNA and RNA molecules. To
translate their advantages to assays for the analysis of proteins with antibodies, new protocols and routines had to be developed and established.
Nowadays, there are multiplexed techniques for measuring the amount of proteins in different sample types (e.g. cells, blood serum, urine), to
determine how proteins are modified in biological processes (e.g. phosphorylation), or to describe specific protein-protein interactions. Another
example is the analysis of antibodies circulating in blood from patients. Microarray-based applications have also been built for purified antibodies
and to study the antibody binding characteristics – an important aspect when using binding reagents as research reagents. Such protein
microarrays can either consist of proteins, protein fragments, or small peptides to test the specificity of the binding reagent. Protein microarrays
can reveal the interactions to entire proteins or larger protein fragments, while peptide microarrays show to which particular parts (epitopes) of the
proteins the antibodies bind. A typical epitope mapping result is shown in Figure 3.20 (Edfors et al., 2014). Synthesizing millions of overlapping
peptides with only one amino acid residue shift on such arrays enables the mapping of antibody binding regions at high resolution. This gives very
detailed information of the linear (continuous) epitopes recognized by an antibody. Just like with proteins, protein fragments or other antigens, the
assembly of peptides on arrays may also be used for studies of antibody reactivity in plasma samples from patients with infectious and autoimmune
diseases.
Figure 3.20. Epitope Mapping of Polyclonal Antibodies. Polyclonal antibodies binding to a peptide array where the result displays four distinct
linear epitopes and the consecutive overlapping peptides which are bound. X-axis: peptides, Y-axis: mean fluorescence intensity (MFI).(Edfors et al.,
2014)
back to the top
Western Blot

Western Blot
Western Blot (WB) is a common method to detect and analyze proteins. It is built on a technique that involves transferring, also known as blotting,
proteins separated by electrophoresis from the gel to a membrane where they can be visualized specifically. The procedure was first described by
H. Towbin et al in 1979(Towbin, Staehelin, & Gordon, 1979) and two years later given its name by W. Neal Burnette(Burnette, 1981). Towbin et al
described electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets where the original gel pattern was accurately
obtained. The setup consists of a standard set of seven steps, Figure 3.21.
Figure 3.21. The Standard Steps in Western Blotting. The standard steps involve sample preparation (1), Gel electrophoresis (2), Blotting to
membrane (3), Antibody probing (4), Detection (5), Imaging (6) and Analysis (7).
Samples are prepared and loaded on to a gel and during the electrophoresis the negatively charged proteins move toward the positively charged
anode. In order to further analyze the proteins, they are transferred onto a membrane in a procedure called blotting. After the transfer, the
membrane is blocked in order to prevent unwanted membrane-protein interaction in the following steps. To visualize the protein of interest the
membrane is commonly first probed using a primary protein-specific antibody followed by a labeled secondary antibody used for detection. An
image is taken of the membrane and the result is analyzed.
By adding a separate marker solution to one of the wells in the gel, it is possible to estimate the size of the protein in addition to the antibody
interactions that are used to verify the specific protein. The separation on the gel is not only due to size but also to some extent depending on the
molecular charge, hydrophobic regions, and degree of denaturation. The setup of the experiment can be varied in many ways to best suit the
specific inquiry. When analyzing the results, variations between lanes regarding loading and transfer rates between blots, must be taken into
consideration. In addition, the non-linear relation of the generated signal across the concentration range of the samples is also an aspect of
consideration when interpreting the results. The outcome of a WB experiment depends on three important factors; the ability of the antibody to
bind a specific protein, the strength of the interaction, and the concentration of the protein of interest itself. Moreover, the specificity of the
binding to the target and a low cross reactivity are important features as well. The result form the WB is not always easy to interpret as the size of
the protein may vary from the theoretical weight due to posttranslational modifications, such as glycosylation, or interactions with other proteins.
However, WB is a very common method and almost all available commercial antibodies have been validatedusing this method.
Sample preparation
The first step of a WB is to prepare the sample, e.g. tissue, cells, or other solution, which is going to be analyzed. Usually the tissue needs to be
broken down by blending, homogenization, or sonication. Buffers are added to lyse the cells and solubilize the proteins and often an inhibitor is
added to prevent denaturation or degradation. Different types of filtration and centrifugation methods are applied to further prepare the samples.
It is important to determine the total protein concentration of the generated extract to be able to load a specific amount on the gel to enable
comparison between samples. Usually a biochemical assay is used in order to determine the protein concentration. The extract is then diluted with
loading buffer consisting of glycerol and a dye (e.g. bromophenol blue). The glycerol is used to simplify the loading by raising the density of the
extract and the dye is added to visualize the sample. Heat is applied on the samples in order to break the structures of the protein, which will help
keeping the negative charge from neutralization(Mahmood & Yang, 2012). Preferably positive and negative controls are included in the set up to
confirm identity of the protein as well as the activity of the antibody.
Gel electrophoresis
After sample preparation the extract is ready to be loaded to separate the proteins according to size by gel electrophoresis.An electric field is
applied over the gel that causes the charged molecules to move. In WB polyacrylamide gels are used for protein separation and the method is
therefore called polyacrylamide gel electrophoresis (PAGE) when using native condition. For denaturing conditions, sodium dodecyl sulfate (SDS) is
added to the system and the method is therefore called SDS-PAGE. The SDS binds to the protein and form a negatively charged micelle around the
protein regardless of inherent charge. The denaturing condition dissolves the tridimensional structure of the proteins and the charge of the protein
becomes relative to its size resulting in separation of the proteins only by size. When using native conditions the mobility is depending on both
charge and hydrodynamic size allowing detection of changes in charge due to chemical degradation, conformational changes due to folding or
unfolding, aggregation, or other binding events.
The gel typically consists of two sections with different densities: (i) a stacking gel, and (ii) a separating gel,(Figure 3.22). The differences between
the two sections are in pH and gel concentration. With somewhat acidic pH and a lower concentration of acrylamide the stacking gel separates the
proteins poorly but allows them to form highly defined sharp bands before they enter the separating gel. With more basic conditions and higher gel
concentration, the separating gel makes the proteins differentiate by size as smaller proteins travel faster in the gel than bigger ones. Precast gels
are convenient; however, it is possible to cast them by hand. The gel is immersed in buffer and the protein samples and markers are loaded to the
wells in the gel. A voltage is applied on the gel and the proteins will start to travel down the gel due to their negative electrical charge. Selecting the
proper voltage is important since too high voltage will overheat the gel and maybe deform the bands.

proper voltage is important since too high voltage will overheat the gel and maybe deform the bands.
Figure 3.22. A Typical Vertical Polyacrylamide Gel in Buffer

Blotting to membrane
After gel electrophoresis the proteins are transferred to a solid support membrane, which is the third step of Western Blot. In the transfer process
voltage is applied to transfer the proteins from the gel to the membrane. The setup includes sponges, filter papers, the gel, and the membrane,
which is placed between the gel and the positive electrode (Figure 3.23). This ensures the migration of the negatively charge proteins from the gel
to the membrane. There are three types of membranes: nitrocellulose, polyvinylidene difluoride (PVDF), and nylon. Even though nylon membranes
are superior in several aspects, the high background binding and irreversible staining of some dyes makes this type of membrane less common than
the other two alternatives. The major advantage of nitrocellulose membranes is the low background regardless of detection method. Due to a
relatively large average pore size, nitrocellulose membranes should not be used for transfer of proteins with low molecular weight. Moreover,
when dry, the membrane becomes brittle which makes them difficult to handle. The more stable PVDF membrane allows relabeling and is more
convenient to store. The hydrophobic nature of PVDF result in high protein binding capacity, however, as a consequence the background is also
higher.
Figure 3.23. Transfer Procedure for Western Blotting. The proteins in the gel are transferred to a membrane and the sample is visualized through
blocking, adding antibodies, and washing according to a certain schedule.
There are two methods for the blotting called wet and semi-dry. The wet conditions are preferred when the transfer must be efficient and give high
quality regarding distinct and sharp bands. In addition, this is the better choice for transfer of larger protein complex. The gel, membrane, and filter
papers are completely immersed in buffer during the transfer and there is no risk of drying out the gel. Semi-dry blotting is more rapid and less

papers are completely immersed in buffer during the transfer and there is no risk of drying out the gel. Semi-dry blotting is more rapid and less
volume of buffer is needed. However, this transfer method is usually less efficient, especially for larger proteins, and there is a risk of overheating
and drying the gel when using extended transfer times.
Antibody probing
The forth step of the WB is antibody probing. In order to prevent unspecific binding of the antibodies to the membrane, rather than binding specific
to the protein of interest, a substance is used to block out the residual sites on the membrane. Common substances used are dried non-fat milk, 5%
Bovine Serum Albumin (BSA) diluted in Tris Buffered Saline Tween (TBST), normal goat serum, casein, or fish gelatin(Mahmood & Yang, 2012). Milk
is easy to get hold of and inexpensive, however not suitable for all detection labels. Fish gelatin gives lower background but can mask some proteins
as well as being a relatively expensive blocking buffer. BSA is inexpensive, whereas serum can contain immunoglobulins giving rise to cross-
reactivity. Careful selection of the blocking agent is key since none of the blocking buffers are ideal for all different antigen-antibody interactions.
The blocking procedure consists of incubating the membrane in the appropriate blocking buffer for an hour or longer. When using long incubation
times, the blocking should be performed at +4°C to rule out the risk of staining artifacts or background. Blocking is a delicate balance between
reducing the background without decreasing the signal from the protein of interest.
The blocked membrane is thereafter incubated with the primary antibody. The antibody is diluted to a suitable concentration in TBST, phosphate
buffered saline (PBS), or wash buffer. It is preferred to incubate the antibody with BSA if the antibody is going to be re-used. After washing the
membrane, the membrane is incubated with the secondary antibody that binds to the primary antibody. The secondary antibody is labeled with a
reporter. When using a polyclonal antibody as secondary antibody, it may give rise to some background. In the case of background staining, the
secondary antibody may be pre-blocked with non-immune serum from the host it was generated in. Optimization of the concentration of the
secondary antibody is recommended due to quite extended variations between antibodies as well as detection system used.
Detection
In the fifth step of a WB, the protein-antibody-antibody complex is detected on the membrane. There are several kinds of labeling of the secondary
antibody, e.g. enzymes, fluorophores, biotinylation, gold-conjugation, and radioisotopes, as exemplified in Figure 3.24. Amongst enzymes the most
common is HRP used together with chemiluminescent, chemifluorescent, or chromogenic substances. HRP has a high substrate specificity giving
low background, is stable, and inexpensive. In chemiluminescense the HRP enzyme catalyzes the oxidation of luminol from the luminol peroxide
detection reagent. The multi-step reaction generates light emission. Certain chemicals like phenols can enhance the emitted light. A direct method
is the use of fluorescence; the fluorophores emit light after being excited and no detection agent is needed. It is well suitable for quantitative
Western and since different fluorophores emit light of different wavelengths it is possible to perform multiplexing and specific detection of more
than one protein at the time. Using a chemical and/or an enzyme to induce the generation of an active fluorophore from a fluorogenic substrate is
called chemifluorescence. To further enhance the signal intensity a two-step biotin streptavidin based system may be used. Gold conjugation is also
a method where proteins stain dark red due to accumulation of gold. It is also possible to use radioisotopes but they require special handling and
are quite expensive.
Figure 3.24. Different Reporter Systems.

Imaging
Imaging is the sixth step of WB and the capturing can be analogue using a film, or digitally preformed with a CCD camera or scanner capturing the
different kinds of emitted signals. The CCD imaging device enables quantitation with high detection sensitivity and a broad linear range with no
chemical waste or need for a dark room. It may be used to detect membranes, stained gels, or for ultraviolet light applications.
Analysis
The last step of a WB is to analyze the results. In a typical qualitative application, the presence of a protein of interest is confirmed, the amount is
approximated by visual inspection, and the size is determined by comparison with a marker. Improvements and developments, especially towards
highly sensitive detection reagents and advanced imaging techniques, make WB a potential tool for quantitative analysis. The quantitative
applications entail a definition of the amount of protein in relative or absolute terms. Some factors are to take under consideration like sensitivity,
signal stability, linear dynamic range, normalization, and the signal-to-noise ratio. The minimum of protein that can be seen in a given assay gives
the limits of detection (LOD), and the limit of signal intensity that can be reliably used for precise quantification is the limit of quantification (LOQ).
Factors that affect these terms are antibody quality and concentrations as well as exposure times when considering the minimum amount of
protein detected. A stable signal system expands the time window for reaching high sensitivity, multiple exposures, and possibility to detect weak
bands. The range that allows an even and precise quantitation where the signal intensity still is proportional to the amount of protein is called the
linear dynamic range. It is important to avoid signal saturation due to excessive amounts of protein or high concentrations of antibodies. A low LOD
and quantitation of both weak and strong signals gives a broad linear dynamic range. The protein of interest should be normalized to an internal
reference that allows fluctuations in amount of protein loaded onto each well or different concentrations. This can be achieved with housekeeping
or spiked protein. The ratio between the signal and noise is important in order to properly quantitate the protein. This is of outmost importance
when detecting weak bands where a higher background is expected. A typical Western Blot is seen in Figure 3.25.

when detecting weak bands where a higher background is expected. A typical Western Blot is seen in Figure 3.25.
Figure 3.25. Typical Western Blot result using HRP-conjugated antibodies and a CCD camera.
back to the top
Immunohistochemistry
Immunohistochemistry (IHC) is a powerful microscopy-based technique for visualizing cellular components, for instance proteins or other
macromolecules in tissue samples. The strength of IHC is the intuitive visual output that reveals the existence and localization of the target-protein
in the context of different cell types, biological states, and/or subcellular localization within complex tissues.
The IHC technique was invented during the 1940s(Coons, Creech, & Jones, 1941) and is routinely used as an important tool in health care and
pathology for e.g. diagnostic purposes or to stratify patients for optimized treatment regimes. IHC is also widely used in research where molecules
of interest are analyzed to study their roles in both healthy and diseased cells and tissues on the molecular, cellular or tissue level. There are many
different ways to perform visualization of targets in tissues using IHC or IHC-based methods, and numerous protocols exist for different applications
and assays. Even though IHC is generally a robust and established method, new assays often need careful optimization depending on the tissue or
on the properties of the target protein, binder-molecule and/or reporter system. Many years of technical development and the hugely increased
availability for specific binding-molecules have greatly improved the usefulness and areas of applications for IHC. The progress in the field of IHC-
based techniques and reagents has enabled scientists and health care providers with more precise tools, assays and biomarkers. In addition,
technical advances have enabled e.g. highly sensitive simultaneous detection of multiple proteins in the same sample, and the detection of protein-
protein interactions.
The classical IHC assay is illustrated in Figure 3.26 and involves detection of epitopes expressed by a single protein-target within a tissue sample
using a “primary antibody” capable of binding those epitopes with high specificity. After the epitope-antibody binding event, a “secondary
antibody” capable of binding the primary antibody with high specificity is added. The secondary antibody is coupled to a reporter molecule and
after the antibody-antibody binding event, a chemical substrate is added which reacts with the reporter molecule to produce a colored precipitate
at the site of the whole epitope-antibody complex.
Figure 3.26. The Basic Principle of Immunohistochemistry. In the schematic illustration, a formalin-fixed paraffin embedded tissue section is
stained using a primary antibody directed towards a specific protein target. A solution containing the primary antibody is added to the tissue
section and the antibodies are allowed some time to find and bind to their target. After this step, unbound and surplus antibodies are washed away
and the secondary antibody is added. The secondary antibody, which carries a linker molecule with horseradish peroxidase (HRP) enzymes, is also
allowed some time to bind to the primary antibody, followed by another washing step. After this, 3,3′ Diaminobenzidine (DAB) is added. The HRP
enzyme transforms the DAB substrate into a brownish precipitate that is deposited in the tissue at the site of the reaction, thus producing a visual
representation of where the primary antibody first bound its target.
Tissue preparation
The tissue plays a central role in the experiment and it is important that it is processed so that epitopes and proper morphology is preserved. The

The tissue plays a central role in the experiment and it is important that it is processed so that epitopes and proper morphology is preserved. The
most common processing for IHC is to prepare formalin-fixed paraffin-embedded (FFPE) tissue blocks. The purpose of formalin fixation is to
produce chemical cross-linking of proteins within the tissue. This terminates all cellular processes and freezes the cellular components at the place
and in the conformation they were in at the time of fixation and also prevent degradation. After adequate fixation, the tissue is further processed
and ultimately embedded in paraffin blocks, which are then sectioned into thin slices (usually 4-10µm) using a microtome. The sections are
transferred to glass slides and allowed to adhere prior to further processing.
Other methods for fixation besides formalin are sometimes used. These include other types of aldehydes or using different alcohol solutions. The
best choice of fixative is very much dependent on the assay. A common alternative to FFPE is to prepare frozen tissue samples. In this case, the
tissue is embedded in a cryoprotective medium and frozen, and fixation is performed post-sectioning. Frozen tissues are sectioned in cryostats and
have the advantage of short processing times and of better preservation of sensitive epitopes, but can often be inferior to FFPE tissues in terms of
preserving histological morphology.
Antigen (epitope) retrieval
A concern associated with cross-linking fixatives like formalin, or too long time spent in fixative medium is the masking of epitopes, which can
obstruct the primary antibody from binding to its target. Especially with FFPE samples, there is often a need to revert some of the chemical
crosslinking and “retrieve” the epitopes before proceeding to the actual IHC. There are several antigen retrieval protocols available and the main
strategies include treating the tissue slide with heat, digestive enzymes, detergents, or combinations thereof. The most common method for
antigen retrieval in FFPE samples is to pressure-boil the tissue slides in an acidic citrate buffer for around 15-20 minutes.
Antibody binding
The quality and specificity of the binding molecule is crucial for any IHC based technique, and the choice of binder can directly affect the outcome,
reliability, and possibly also the interpretation of the assay. Antibodies are by far the most common type of binding-molecule used for IHC, and
although most antibodies are able to adequately detect the correct molecule of interest, they may also vary greatly in their specificity for their
intended target. Antibodies with high specificity are therefore more reliable when interpreting “on-target” binding, since they produce little or no
“off-target” binding or “background”. Antibodies that are less specific can produce more off-target binding, and the resulting background will
possibly interfere with the correct interpretation of the true on-target signals. There are two main types of antibodies; polyclonal antibodies which
is a heterogeneous mix of antibodies that bind different epitopes on the target and monoclonal antibodies that all bind the same epitope.
Polyclonal antibodies are often very potent due to their ability to detect and bind multiple epitopes on the same target. However, the epitopes they
bind are often poorly defined and with multiple and varying epitope-specificities comes the increased likelihood of off-target binding events and
background noise. However, the potency of polyclonal antibodies can be advantageous since the concentration of binding events around the on-
target molecule usually outweighs potential background noise. A drawback is that polyclonal antibodies are usually limited resources since they are
derived from animal sera. Monoclonal antibodies, by contrast, have more continuity since they can be produced in hybridoma cell lines.
Monoclonal antibodies are also often well defined in terms of epitope binding, but can still generate results that are hard to interpret if the
specificity is low or if the target epitope is present in low abundance.
Careful optimization and titration of antibody concentration for each assay is needed, since the result is dependent not only on the antibody’s
specificity and affinity for the target, but also on the concentration and availability of on-target and potential off-target epitopes present in the
sample. Adding too much antibodies to the sample will increase the number of possible low-affinity off-target binding events once the on-target
epitope(s) are saturated with binders. By lowering the antibody concentration, off-target binding events become rarer as they usually have lower
affinity than on-target binding events. The risk when attempting to reduce background while using a low-affinity antibody is that the on-target
signals are concomitantly weakened to the point of providing a false negative result.
Other types of binder molecules sometimes used in IHC-based techniques include affibodies, peptides, antibody fragments or other small
molecules.
Detection systems
The whole purpose of performing IHC is to obtain a visual representation of where the target can be found within the experimental tissue, and
preferably also gain information about the target’s expression pattern among heterogeneous cell populations and/or subcellular localizations. This
is exemplified in Figure 3.27, which illustrates how different antibodies are used to visualize different cellular or tissue compartments within a
complex tissue. To visualize the target-antibody interaction, some kind of detection system that produces an observable stain or signal is needed.
The most common method for introducing a detection system to the experiment is to use a secondary antibody that carries a pre-bound reporter
molecule, i.e. enzyme or fluorophore. Secondary antibodies are usually targeted specifically towards antibody molecules from a different animal
species. For example, if the primary antibody is raised in a rabbit, then the secondary antibody must be raised in another animal and targeted
specifically towards rabbit antibodies.

specifically towards rabbit antibodies.
Figure 3.27. Visualizing different protein targets in complex tissues. The right column shows a magnification of the corresponding images in the
left column. In the IHC image, consecutive sections of human esophagus stained using four different antibodies allows for direct comparison of
different protein expression patterns within the tissue and within subcellular compartments. The top images are only counterstained for
hematoxylin for comparison. The p63 antibody stains cell nuclei in a population of cells that reside in the basal part of the esophageal epithelium.
The EGFR (Epidermal growth factor receptor) antibody appears to stain the same cell population as p63, but stains cellular membranes instead of
nuclei. The G6PD (Glucose-6-phosphate dehydrogenase) antibody stains the cytoplasm of a wider repertoire of esophageal epithelial cells and also
cells residing in the connective tissue. The Laminin (LAMB2) antibody stains only cells and structures in the connective tissue underlying the
esophagus.
For FFPE tissue samples the most common detection method is to use enzymatic reactions to generate a colored precipitate at the site of antibody
binding. The secondary antibodies then carry an enzyme, e.g. horseradish peroxidase (HRP) or alkaline phosphatase (AP), that are capable of
converting chromogens like 3,3′ Diaminobenzidine (DAB) or 5-bromo-4-chloro-3-indolyl phosphate/ p-nitroblue tetrazolium chloride (BCIP/NBT)
into brown or bluish precipitates that are deposited in the tissue at the site of the reaction. Chromogenic stains are observable in light-microscopy
and are usually very stable over long periods of time, which is beneficial if the experiment needs to be archived or reviewedat a later time point.
For frozen tissue sections it is more common to use fluorophore-linked secondary antibodies that emit a specific color (usually green, red, or blue)
when excited by the correct wavelengths of light. Moreover, fluorophores are usually not stable for long periods of time. However, the benefit of
using fluorophores is that they provide an easy method for performing double-labeling experiments where several antibodies towards multiple
targets are assayed in the same sample. The secondary antibodies need to be targeted towards different primary antibodies and also to be coupled

targets are assayed in the same sample. The secondary antibodies need to be targeted towards different primary antibodies and also to be coupled
to different fluorophores. The different secondary antibodies are then observed separately by exciting them sequentially with different
wavelengths of light. These different excitation results are saved as separate images (or color channels) and may later be overlaid to infer protein
co-localizations etc.
Using reporter-carrying secondary antibodies for detection is in itself an amplification step since several secondary antibodies are able to bind a
single primary antibody, but sometimes further amplification steps are desired to increase the signal and sensitivity of the experiment. In such
cases, the secondary antibody may instead carry “linker molecules”, for instance biotin polymers, which are able to recruit a larger number of
reporter molecules in subsequent steps. This strategy for amplifying signals is useful for both enzymatic and fluorescent detection methods.
Counterstaining
Immunohistochemical staining using chromogens often benefits from having a counterstain applied that enhances the contrast and facilitates the
observation of histological features. The most common type of counterstain used for FFPE samples is hematoxylin that stains cellular cytoplasm
with a pale bluish color, and stain cell nuclei in a darker bluish nuance, as shown in Figure 3.26. Fluorescent stainings are usually not counterstained
with hematoxylin, since the detection method is not based on light microscopy. Instead, the most common way to obtain counterstaining for
fluorescence is to label cell nuclei by adding fluorescent dyes that bind nucleic acids, as shown in Figure 3.28. After the actual immunohistochemical
reaction, the only remaining steps are to coverslip and seal the sample for protection and longterm storage. The most common way is to “glue” the
coverslip to the sample using commercially available purpose-made resins.
Figure 3.28 Endothelial cells under the microscope.Nuclei are stained blue with DAPI, microtubles are marked green by an antibody bound to FITC
and actin filaments are labelled red with phalloidin bound to TRITC. Bovine pulmonary artery endothelial cells
Image from NIH ImageJ-Programmpaket
Specific examples
IHC is widely used in both research and clinical practice.The Human Protein Atlas (HPA) projectis a prime example of how high-throughput IHC is
used to achieve large-scale mapping of the human proteome in a multitude of tissues, cancers and cells. In the HPA project, a streamlined in-house
large scale antibody production chain facilitates the generation of specific antibodies, which after passing basic characterization and validation
regimes, are used to systematically stain tissue microarrays containing hundreds of tissue cores within a single experiment. The system for IHC
employed by HPA relies heavily on standardization of protocols and automatisation using machines, but the evaluation of the optimal titration for
each antibody is performed manually before the antibody is approved for staining on the full set of tissues. Each stained tissue core is annotated
with respect to immunohistochemical staining in tissues and cell types, and thereafter published as a high resolution image on the web portal to be
freely viewed by anyone.
In clinical practice, IHC is mainly used within pathology to aid physicians to evaluate tissue specimens with respect to healthy and or diseased
states, to set diagnoses, and to define the molecular subtype of different types of cancer. A specific example where IHC is used diagnostically is
when pathologists are presented with a metastatic tumor sample and the tissue origin of the primary tumor is unknown. In these cases,
pathologists use a panel of different antibodies that target tissue specific proteins, such as prostate-specific antigen for prostate cancer, or estrogen
receptor for gynecological cancers, or cytokeratin 20 for gastrointestinal cancers(Gremel et al., 2014). Once a broad classification is made,
additional tissue specific antibodies are used to further pinpoint the origin of the primary tumor. This information is useful for choosing the best or
most appropriate strategy for drug therapy and/or to locate the primary tumor for radiation therapy and/or surgery.
back to the top

Solid-Phase Protein Synthesis
Peptides are chemically synthesized by the condensation reaction of the carboxyl group of one amino acid to the amino group of another.
Protecting group strategies are usually necessary to prevent undesirable side reactions with the various amino acid side chains. Chemical peptide
synthesis most commonly starts at the carboxyl end of the peptide (C-terminus), and proceeds toward the amino-terminus (N-terminus). Protein
biosynthesis (long peptides) in living organisms occurs in the opposite direction. Chemical synthesis facilitates the production of peptides which are
difficult to express in bacteria, the incorporation of unnatural amino acids, peptide/protein backbone modification, and the synthesis of D-proteins,

difficult to express in bacteria, the incorporation of unnatural amino acids, peptide/protein backbone modification, and the synthesis of D-proteins,
which consist of D-amino acids.
The established method for the production of synthetic peptides in the lab is known as solid-phase peptide synthesis (SPPS). Pioneered by Robert
Bruce Merrifield, SPPS allows the rapid assembly of a peptide chain through successive reactions of amino acid derivatives on an insoluble porous
support.
The solid support consists of small, polymeric resin beads functionalized with reactive groups (such as amine or hydroxyl groups) that link to the
nascent peptide chain. Since the peptide remains covalently attached to the support throughout the synthesis, excess reagents and side products
can be removed by washing and filtration. This approach circumvents the comparatively time-consuming isolation of the product peptide from
solution after each reaction step, which would be required when using conventional solution-phase synthesis.
Each amino acid to be coupled to the peptide chain N-terminus must be protected on its N-terminus and side chain using appropriate protecting
groups such as Boc (acid-labile) or Fmoc (base-labile), depending on the side chain and the protection strategy used (FIg. 3.29).
The general SPPS procedure is one of repeated cycles of alternate N-terminal deprotection and coupling reactions. The resin can be washed
between each steps. First an amino acid is coupled to the resin. Subsequently, the amine is deprotected, and then coupled with the free acid of the
second amino acid. This cycle repeats until the desired sequence has been synthesized. SPPS cycles may also include capping steps which block the
ends of unreacted amino acids from reacting. At the end of the synthesis, the crude peptide is cleaved from the solid support while simultaneously
removing all protecting groups using a reagent strong acids like trifluoroacetic acid or a nucleophile. The crude peptide can be precipitated from a
non-polar solvent like diethyl ether in order to remove organic soluble by products. The crude peptide can be purified using reversed-phase HPLC.
The purification process, especially of longer peptides can be challenging, because small amounts of several byproducts, which are very similar to
the product, have to be removed. For this reason so-called continuous chromatography processes such as MCSGP are increasingly being used in
commercialsettings to maximize the yield without sacrificing on purity levels.
SPPS is limited by reaction yields, and typically peptides and proteins in the range of 70 amino acids are pushing the limits of synthetic accessibility.
Synthetic difficulty also is sequence dependent; typically aggregation-prone sequences such as amyloids are difficult to make. Longer lengths can be
accessed by using ligation approaches such as native chemical ligation, where two shorter fully deprotected synthetic peptides can be joined
together in solution.
Figure 3.29 Solid-phase synthesis of a dipeptide using an (amine-functionalized) amide resin. The N-terminal protecting group (PG) can be Fmoc
or Boc, depending on the protecting group scheme used. The amino acid side chains (R1, R2 etc.) are orthogonally protected.
Image by Bédard, F. and Biron, E. (2018) Frontiers in Microbiology 9:1048
Protein Sequencing using Edman Degradation

Edman degradation, developed by Pehr Edman, is a method of sequencing amino acids in a peptide (Fig. 3.30). In this method, the amino-terminal
residue is labeled and cleaved from the peptide without disrupting the peptide bonds between other amino acid residues.
Figure 3.30 Edman Degradation Scheme. In this method, Phenyl isothiocyanate is reacted with an uncharged N-terminal amino group, under mildly
alkaline conditions, to form a cyclical phenylthiocarbamoyl derivative. Then, under acidic conditions, this derivative of the terminal amino acid is
cleaved as a thiazolinone derivative. The thiazolinone amino acid is then selectively extracted into an organic solvent and treated with acid to form
the more stable phenylthiohydantoin (PTH)- amino acid derivative that can be identified by using chromatography or electrophoresis. This
procedure can then be repeated again to identify the next amino acid.
Image fromChoij
A major drawback to Edman degradation is that the peptides being sequenced in this manner cannot have more than 50 to 60 residues (and in
practice, under 30). The peptide length is limited due to the cyclical derivatization not always going to completion. The derivatization problem can

practice, under 30). The peptide length is limited due to the cyclical derivatization not always going to completion. The derivatization problem can
be resolved by cleaving large peptides into smaller peptides before proceeding with the reaction. It is able to accurately sequence up to 30 amino
acids with modern machines capable of over 99% efficiency per amino acid. An advantage of the Edman degradation is that it only uses 10 – 100
pico-moles of peptide for the sequencing process. The Edman degradation reaction was automated in 1967 by Edman and Beggs to speed up the
process and 100 automated devices were in use worldwide by 1973.
Because the Edman degradation proceeds from the N-terminus of the protein, it will not work if the N-terminus has been chemically modified (e.g.
by acetylation or formation of pyroglutamic acid). Sequencing will stop if a non-α-amino acid is encountered (e.g. isoaspartic acid), since the
favored five-membered ring intermediate is unable to be formed. Edman degradation is generally not useful to determine the positions of disulfide
bridges. It also requires peptide amounts of 1 picomole or above for discernible results.
Following 2D SDS PAGE the proteins can be transferred to a polyvinylidene difluoride (PVDF) blotting membrane for further analysis. Edman
degradations can be performed directly from a PVDF membrane. N-terminal residue sequencing resulting in five to ten amino acid may be sufficient
to identify a Protein of Interest (POI).
Sequence analysis by Matrix-Assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF) Mass Spectrometry.
Mass spectrometry is a technique to analyze with high accuracy the composition of different chemical elements and atomic isotopes splitting their
atomic nuclei according to their mass- charge ratio (m/z). It can be used to identify different chemical elements that form a compound or to
determine the isotopic content of different elements in the same compound.
Protein mass spectrometry refers to the application of mass spectrometry to the study of proteins. Mass spectrometry is an important method for
the accurate mass determination and characterization of proteins, and a variety of methods and instrumentations have been developed for its
many uses. Its applications include the identification of proteins and their post-translational modifications, the elucidation of protein complexes,
their subunits and functional interactions, as well as the global measurement of proteins in proteomics. It can also be used to localize proteins to
the various organelles, and determine the interactions between different proteins as well as with membrane lipids.
Two techniques are often used with liquid and solid biological samples: electro spray ionization and laser matrix- assisted laser
desorption/ionization (MALDI). In the MALDI ionization analytes co- crystallized with a suitable matrix are converted into ions by the action of a
laser. This source of ionization is usually associated with a time of flight analyzer (TOF) in which the ions are separated according to their mass-
charge after being accelerated in an electric field. At last, a mass spectrometer detector records the charge induced or current produced when an
ion passes by or hits a surface. A mass spectrum is recorded for each protein (Figure 3.31).
Figure 3.31. Protein identification by MALDI-TOF MS. Workflow of protein identification developing MALDI-TOF MS assay (A), followed by MS/MS
fragmentation of peptides (B) and analysis of spectral data with the MASCOT database search algorithm (C).
In general, proteins are analyzed either in a “top-down” approach in which proteins are analyzed intact, or a “bottom-up” approach in which
protein are first digested into fragments. An intermediate “middle-down” approach in which larger peptide fragments are analyzed may also
sometimes be used. The top-down approach however is mostly limited to low-throughput single-protein studies due to issues involved in handling
whole proteins, their heterogeneity and the complexity of their analyses.
In the second approach, referred to as the “bottom-up” MS, proteins are enzymatically digested into smaller peptides using a protease such as
trypsin. Trypsin is a serine protease from the PA clan superfamily, found in the digestive system of many vertebrates, where it hydrolyzes proteins.
Trypsin cleaves peptide chains mainly at the carboxyl side of the amino acids lysine or arginine, except when either is followed by proline. It is used
for numerous biotechnological processes. The process is commonly referred to as trypsin proteolysis or trypsinisation, and proteins that have been
digested/treated with trypsin are said to have been trypsinized.
Subsequently, these peptides are introduced into the mass spectrometer and identified by peptide mass fingerprinting or tandem mass
spectrometry. Hence, this approach uses identification at the peptide level to infer the existence of proteins pieced back together with de novo
repeat detection. The smaller and more uniform fragments are easier to analyze than intact proteins and can be also determined with high
accuracy, this “bottom-up” approach is therefore the preferred method of studies in proteomics. A further approach that is beginning to be useful
is the intermediate “middle-down” approach in which proteolytic peptides larger than the typical tryptic peptides are analyzed.
Proteins of interest are usually part of a complex mixture of multiple proteins and molecules, which co-exist in the biological medium. This presents
two significant problems. First, the two ionization techniques used for large molecules only work well when the mixture contains roughly equal
amounts of constituents, while in biological samples, different proteins tend to be present in widely differing amounts. If such a mixture is ionized
using electrospray or MALDI, the more abundant species have a tendency to “drown” or suppress signals from less abundant ones. Second, mass
spectrum from a complex mixture is very difficult to interpret due to the overwhelming number of mixture components. This is exacerbated by the
fact that enzymatic digestion of a protein gives rise to a large number of peptide products.
In light of these problems, the methods of one- and two-dimensional gel electrophoresis and high performance liquid chromatography are widely
used for separation of proteins. The first method fractionates whole proteins via two-dimensional gel electrophoresis (Figure 3.32). The first-
dimension of 2D gel is isoelectric focusing (IEF). In this dimension, the protein is separated by its isoelectric point (pI) and the second-dimension is
SDS-polyacrylamide gel electrophoresis (SDS-PAGE). This dimension separates the protein according to its molecular weight. Once this step is
completed in-gel digestion occurs.
In some situations, it may be necessary to combine both of these techniques. Gel spots identified on a 2D Gel are usually attributable to one
protein. If the identity of the protein is desired, usually the method of in-gel digestion is applied, where the protein spot of interest is excised, and
digested proteolytically. The peptidemasses resulting from the digestion can be determined by mass spectrometry using peptide mass
fingerprinting. If this information does not allow unequivocal identification of the protein, its peptides can be subject to tandem mass spectrometry
for de novo sequencing. Small changes in mass and charge can be detected with 2D-PAGE. The disadvantages with this technique are its small
dynamic range compared to other methods, some proteins are still difficult to separate due to their acidity, basicity, hydrophobicity, and size (too
large or too small).
The second method, high performance liquid chromatography is used to fractionate peptides after enzymatic digestion. Characterization of protein
mixtures using HPLC/MS is also called shotgun proteomics and MuDPIT (Multi-Dimensional Protein Identification Technology). A peptide mixture

mixtures using HPLC/MS is also called shotgun proteomics and MuDPIT (Multi-Dimensional Protein Identification Technology). A peptide mixture
that results from digestion of a protein mixture is fractionated by one or two steps of liquid chromatography. The eluent from the chromatography
stage can be either directly introduced to the mass spectrometer through electrospray ionization, or laid down on a series of small spots for later
mass analysis using MALDI.
Figure 3.32 Schematic of Protein Fingerprinting by Mass Spectrometry. Protein mixtures are prepared from cell culture or tussie samples and
separated by gel electrophoresis. Single proteins are isolated and digested using trypsin to produce a peptide mixture. Peptides are separated by
liquid chromatography and analyzed by mass spectrometry
Image by Philippe Hupé
back to the top

X-Ray Crystallography
Protein X-Ray Crystallography is a technique used to obtain the three-dimensional structure of a particular protein by X-ray diffraction of its
crystallized form. This three dimensional structure is crucial to determining a protein’s functionality. Making crystals creates a lattice in which this
technique aligns millions of proteins molecules together to make the data collection more sensitive. It’s like getting a stack of papers, measuring the
width with a ruler, and dividing that length with the number of pages to determine the width of one piece of paper. By this averaging technique,
the noise level gets reduced and the signal to noise ratio increases. The specificity of the protein’s active sites and binding sites is completely
dependent on the protein’s precise conformation. In addition to protein structure, other biological molecules can be investigated using X-ray
crystallography. It was the X-ray crystallography by Rosalind E.Franklin, that made it possible for J.D. Watson and F.H.C. Crick to figure out the
double-helix structure of DNA.
X-ray crystallography can reveal the detailed three-dimensional structures of thousands of proteins. The three components in an X-ray
crystallographic analysis are a protein crystal, a source of X-rays, and a detector.
X-ray crystallography is used to investigate molecular structures through the growth of solid crystals of the molecules they study. Crystallographers
aim high-powered X-rays at a tiny crystal containing trillions of identical molecules. The crystal scatters the X-rays onto an electronic detector. The
electronic detector is thesame type used to capture images in a digital camera. After each blast of X-rays, lasting from a few seconds to several
hours, the researchers precisely rotate the crystal by entering its desired orientation into the computer that controls the X-ray apparatus. This
enables the scientists to capture in three dimensions how the crystal scatters, or diffracts, X-rays. The intensity of each diffracted ray is fed into a
computer, which uses a mathematical equation to calculate the position of every atom in the crystallized molecule. The result is a three-
dimensional digital image of the molecule (Figure 3.33).
Crystallographers measure the distances between atoms in angstroms. The perfect “rulers” to measure angstrom distances are X-rays. The X-rays
used by crystallographers are approximately 0.5 to 1.5 angstroms long, which are just the right size to measure the distance between atoms in a
molecule. If the radiation had a wavelength much bigger or much smaller than the bond length of a covalent bond, the light would not diffract and
no new knowledge of the structure would be obtained. That is why X-rays are used.

Figure 3.33 Workflow for Solving the Structure of a Molecule by X-ray Crystallography
Image fromThomas Splettstoesser
The process begins by crystallizing a protein of interest. Crystallization of protein causes all the protein atoms to be orientated in a fixed way with
respect to one another while still maintaining their biologically active conformations – a requirement for X-ray diffraction. A protein must be
precipitated out or extracted from a solution. The rule of thumb here is to get as pure a protein as possible to grow lots of crystals (this allows for
the crystals to have charged properties, and surface charged distribution for better scattering results). 4 critical steps are taken to achieve protein
crystallization, they are:
1. Purify the protein. Determine the purity of the protein and if not pure (usually >99%), then must undergo further purification.
2. Must precipitate protein. Usually done so by dissolving the protein in an appropriate solvent(water-buffer soln. w/ organic salt such as 2-
methyl-2,4-pentanediol). If protein is insoluble in water-buffer or water-organic buffer then a detergent such as sodium lauryl sulfate must be
added.
3. The solution has to be brought to supersaturation(condensing the protein from the rest of the solvent forming condensation nuclei). This is
done by adding a salt to the concentrated solution of the protein, reducing its solubility and allowing the protein to form a highly organized
crystal (this process is referred to as salting out). Other methods include batch crystallization, liquid-liquid crystallization, vapor diffusion, and
dialysis.
4. Let the actual crystals grow. Since nuclei crystals are formed this will lead to obtaining actual crystal growth.
The crystals are then bombarded with X-rays, the diffraction patterns recorded, and the structure is reconfigured from the diffraction pattern using
Fourier Transformation. Through the Fourier Transform, the electron density distribution is illustrated as a series of parallel shapes and lines
stacked on top of each other (contour lines), like a terrain map. The mapping gives a three-dimensional representation of the electron densities
observed through the x-ray crystallography. When interpreting the electron density map, resolution needs to be taken into account. A resolution of
5Å – 10Å can reveal the structure of polypeptide chains, 3Å – 4Å of groups of atoms, and 1Å – 1.5Å of individual atoms. The resolution is limited by
the structure of the crystal and for proteins is about 2Å.
Many advances in drug discovery and medicine are due in large part by X-Ray Crystallography by identifying drug targets in many diseases that
thrive today. In the late 80’s for example, scientists made a breakthrough in using X-Ray Crystallography to produce the structure of HIV Protease,
an enzyme that was vital to the retrovirus’ life cycle. The enzyme cuts viral proteins strands that are main components of immature viral cells into
separate, mature proteins that can continue on to form more mature and infectious viral particles. By looking closely at it structure, specifically its
symmetry, researchers began making compounds that interacted with the active site of the enzyme, which is in the middle of its symmetric halves,
to shut the enzyme down and prevent it from functioning properly. Amazingly, by the mid 90s, three HIV Protease inhibitor drugs were on the
market, drastically reducing the death rate of the AIDS Virus (Figure 3.34)

Figure 3.34. Crystal structure of the D-enantiomer of backbone engineered HIV-1 PR prepared by totalchemical synthesis complexed to D-
MVT101 inhibitor. (a) Cocrystals were obtained from50% ammonium sulfate, pH 5.4, in the space group P212121a = 67.5, b = 92.8, c = 29.4 Å.There
were two monomers of protein and one inhibitor in the crystallographic asymmetricunit. (b) Stereo view of Cα tracing of D HIV-1 PR. Bound D
MVT-101 inhibitor is shown asgreen sticks. Coordinates were deposited to HIV Structural Database;48 accession code:NCI2009
Image from:Miller, M. (2010) Biopolymers 94(4):521-529.
back to the top
Nuclear Magnetic Resonance (NMR) Imaging

Nuclear magnetic resonance spectroscopy of proteins (usually abbreviated protein NMR) is a field of structural biology in which NMR spectroscopy
is used to obtain information about the structure and dynamics of proteins, and also nucleic acids, and their complexes. The field was pioneered by
Richard R. Ernst and Kurt Wüthrich at the ETH, and by Ad Bax, Marius Clore, and Angela Gronenborn at the NIH, among others. Structure
determination by NMR spectroscopy usually consists of several phases, each using a separate set of highly specialized techniques. The sample is
prepared, measurements are made, interpretive approaches are applied, and a structure is calculated and validated.
NMR involves the quantum mechanical properties of the central core (“nucleus”) of the atom. These properties depend on the local molecular
environment, and their measurement provides a map of how the atoms are linked chemically, how close they are in space, and how rapidly they
move with respect to each other. These properties are fundamentally the same as those used in the more familiar magnetic resonance imaging
(MRI), but the molecular applications use a somewhat different approach, appropriate to the change of scale from millimeters (of interest to
radiologists) to nanometers (bonded atoms are typically a fraction of a nanometer apart). This change of scale requires much higher sensitivity of
detection and stability for long term measurement. In contrast to MRI, structural biology studies do not directly generate an image, but rely on
complex computer calculations to generate three-dimensional molecular models.
Currently most samples are examined in a solution in water, but methods are being developed to also work with solid samples. Data collection
relies on placing the sample inside a powerful magnet, sending radio frequency signals through the sample, and measuring the absorption of those
signals. Depending on the environment of atoms within the protein, the nuclei of individual atoms will absorb different frequencies of radio signals.
Furthermore, the absorption signals of different nuclei may be perturbed by adjacent nuclei. This information can be used to determine the
distance between nuclei. These distances in turn can be used to determine the overall structure of the protein.
A typical study might involve how two proteins interact with each other, possibly with a view to developing small molecules that can be used to
probe the normal biology of the interaction (“chemical biology”) or to provide possible leads for pharmaceutical use (drug development).
Frequently, the interacting pair of proteins may have been identified by studies of human genetics, indicating the interaction can be disrupted by
unfavorable mutations, or they may play a key role in the normal biology of a “model” organism like the fruit fly, yeast, the worm C. elegans, or
mice.
Protein nuclear magnetic resonance is performed on aqueous samples of highly purified protein. Usually, the sample consists of between 300 and
600 microlitres with a protein concentration in the range 0.1 – 3 millimolar. The source of the protein can be either natural or produced in a
production system using recombinant DNA techniques through genetic engineering. Recombinantly expressed proteins are usually easier to
produce in sufficient quantity, and this method makes isotopic labeling possible.
The purified protein is usually dissolved in a buffer solution and adjusted to the desired solvent conditions. The NMR sample is prepared in a thin-
walled glass tube. (Figure 3.35)

Figure 3.35 The NMR Sample is Prepared in a Thin-Walled Glass Tube
Image fromKjaergaard
With unlabelled protein the usual procedure is to record a set of two dimensional homonuclear nuclear magnetic resonance experiments through
correlation spectroscopy (COSY), of which several types include conventional correlation spectroscopy, total correlation spectroscopy (TOCSY) and
nuclear Overhauser effect spectroscopy (NOESY). A two-dimensional nuclear magnetic resonance experiment produces a two-dimensional
spectrum. The units of both axes are chemical shifts. The COSY and TOCSY transfer magnetization through the chemical bonds between adjacent
protons (Figure 3.36). The conventional correlation spectroscopy experiment is only able to transfer magnetization between protons on adjacent
atoms, whereas in the total correlation spectroscopy experiment the protons are able to relay the magnetization, so it is transferred among all the
protons that are connected by adjacent atoms. Thus in a conventional correlation spectroscopy, an alpha proton transfers magnetization to the
beta protons, the beta protons transfers to the alpha and gamma protons, if any are present, then the gamma proton transfers to the beta and the
delta protons, and the process continues. In total correlation spectroscopy, the alpha and all the other protons are able to transfer magnetization to
the beta, gamma, delta, epsilon if they are connected by a continuous chain of protons. The continuous chain of protons are the sidechain of the
individual amino acids.
Thus these two experiments are used to build so called spin systems, that is build a list of resonances of the chemical shift of the peptide proton,
the alpha protons and all the protons from each residue’s sidechain. Which chemical shifts corresponds to which nuclei in the spin system is
determined by the conventional correlation spectroscopy connectivities and the fact that different types of protons have characteristic chemical
shifts. To connect the different spinsystems in a sequential order, the nuclear Overhauser effect spectroscopy experiment has to be used. Because
this experiment transfers magnetization through space, it will show crosspeaks for all protons that are close in space regardless of whether they are
in the same spin system or not. The neighbouring residues are inherently close in space, so the assignments can be made by the peaks in the NOESY
with other spin systems.

with other spin systems.
Figure 3.36: Comparison of a COSY and TOCSY 2D spectra for an amino acid like glutamate or methionine. The TOCSY shows off diagonal
crosspeaks between all protons in the spectrum, but the COSY only has crosspeaks between neighbours.
Image from Kjaergaard
One important problem using homonuclear nuclear magnetic resonance is overlap between peaks. This occurs when different protons have the
same or very similar chemical shifts. This problem becomes greater as the protein becomes larger, so homonuclear nuclear magnetic resonance is
usually restricted to small proteins or peptides.
If recombinant proteins can be produced, the resulting protein can be labelled with Nitrogen-15 or with Carbon-13 to allow for more detailed
experimentation, such as heteronuclear single quantum coherence spectroscopy (HSQC). The most commonly performed 15N experiment is the
1 15
H- N HSQC. The experiment is highly sensitive and therefore can be performed relatively quickly. It is often used to check the suitability of a
protein for structure determination using NMR, as well as for the optimization of the sample conditions. It is one of the standard suite of
experiments used for the determination of the solution structure of protein. The HSQC can be further expanded into three- and four dimensional
NMR experiments, such as 15N-TOCSY-HSQC and 15N-NOESY-HSQC (Figure 3.37).
1
H–15N HSQC spectrum of a fragment of an isotopically labeled protein NleG3-2. Each peak in the spectrum represents a bonded N-H pair, with its
two coordinates corresponding to the chemical shifts of each of the H and N atoms. 1H–15N HSQC spectrum of a fragment of an isotopically labeled
protein NleG3-2. Each peak in the spectrum represents a bonded N-H pair, with its two coordinates corresponding to the chemical shifts of each of
the H and N atoms.

Figure 3.37 1H–15N HSQC spectrum of a fragment of an isotopically labeled protein NleG3-2. Each peak in the spectrum represents a bonded N-H
pair, with its two coordinates corresponding to the chemical shifts of each of the H and N atoms.
Image from:Wu, B., et. al. (2010) PLoS Pathogens 6(6):e1000960.
Cryo-electron Microscopy
Cryogenic-electron microscopy (cryo-EM) has recently emerged as a powerful technique in structural biology that is capable of delivering high-
resolution density maps of macromolecular structures (Fig. 3.38). Resolutions approaching 1.5 Å are now possible and maps in the 1–4-Å range
inform the construction of atomistic models with a high degree of confidence. This new capacity for investigators to determine macromolecular
structures at high resolution and without the need for crystallogenesis has led to an explosion of interest in adopting cryo-EM.
Protein suspensions are frozen on 3-mm-diameter transmission-electron microscope (TEM) support grids made from a conductive material (e.g. Cu
or Au) that are coated with a carbon film with a regular array of perforations 1–2 μm in diameter. A total of 3–5 μl of sample is loaded onto the grid
which is then immediately blotted with filter paper with the aim of creating a film of buffer/protein on the grid that, when frozen, will be thin
enough for the electron beam to penetrate. Optimising the ice thickness is a vital step in sample preparation as thicker layers of ice increase the
probability that the incident electron will undergo multiple scattering events and thereby reduce the image quality. In the case of extreme ice
thickness, the electron beam does not penetrate at all. After blotting, the grid is rapidly plunged into a bath of liquid ethane—a very effective
cryogen that freezes water with sufficient rapidity as to prevent formation of ice crystals. The formation of a vitreous layer of ice is the fundamental
step in cryo-EM and preserves the target in a near-native state. The resulting vitreous ice layer with suspended protein molecules must then remain
close to liquid nitrogen temperature (− 196 °C) during storage and imaging in the TEM to prevent phase changes to other types of ice that are not
amenable to high-quality imaging and preservation of protein structure.
Image formation in cryo-EM is primarily by phase contrast, although between 7 and 10% of image contrast is from amplitude contrast. Amplitude
contrast, where an electron is scattered to such an extent it is removed by an aperture or energy filter along the path of the TEM column, is
generally not considered, as the information obtained is usually low resolution compared with phase contrast.
Figure 3.38 Cryo-Electron Microscopy. (a) the Scottish Centrel for Macromolecular Imaging JEOL CryoARM 300. (b) High-resolution 2.2-Å resolution
structure of lumazine synthase.
Figure from: Bhella, D. (2019) Biophysical Reviews 11:515-519
Advantages and Disadvantages to Structure Elucidation

Obtaining a pure, highly concentrated (mM) protein sample is a major bottleneck for both x-ray crystallography and NMR. The high concentration is
required because both techniques are insensitive to single molecule analysis, and a large population of a particular protein is required to overcome
the signal-to-noise barrier. On a similar note the sample needs to be very homogenous, so protein purification is necessary at some point. Cryo-EM
requires considerably less protein than for the other two methods, but still ‘a lot’ by any standard. Typically, Cryo-EM requires preparations at a
concentration of 1 mg/ml in a volume of at least 50 μl, whereas crystallogenesis migh require 500 μlof protein at a concentration of 5 -10 mg/ml.
Cryo-Em also requires the protein to be prepared in a low-salt buffer, with minimal additives, to ensure good freezing and image contrast.
Recombinant protein production using E. coli is the method of choice when large quantities of protein are required. This process involves taking the
gene (often cDNA) of the protein of interest, splicing it into a suitable inducible vector, transforming the vector into an E. coli host, and growing the
culture in a rich medium. The bacterial host will multiply during a growth phase, after which it is induced to express the protein of interest. If all
goes well, the protein will express solubly and in high numbers. Unfortunately, this process is easier saidthan done. Many eukaryotic proteins do
not express well in prokaryotic hosts, and oftentimes modifications need to be made to optimize the bacterial host, codon usage, media, etc. to
obtain a decent yield of recombinant protein. Additionally, proteins often express insolubly as inclusion bodies and require high concentrations (2M
to 8M) of denaturants such as urea or guanidine hydrochloride to solubilize them, and then stepwise dialysis into an appropriate buffer to refold
them. Alternatively, eukaryotic organisms such as S. cerevisiae (yeast), insect and mammalian cell lines can be used, especially when post-
translation modifications are required, though a decrease in yield and increase in overall cost is common with these organisms.
The difficulty of protein production is compounded for NMR by the fact that all proteins need to be 15N and/or 13C labeled, as only these isotopes

The difficulty of protein production is compounded for NMR by the fact that all proteins need to be 15N and/or 13C labeled, as only these isotopes
have nuclei with + ½ and – ½ spin states which enable the energy transitions required for a radiofrequency NMR signal; note that 1H also has ½ spin
states but is highly abundant.
Protein stability is an issue for both crystallography and NMR. Once a protein has been expressed, purified, and concentrated, it must maintain its
structural integrity for the duration of the experiments. For crystallography, this involves the crystallization process, where the protein sample is
placed in a variety of solutions (most often involving high concentrations of polyethylene glycol) that induce crystallization. Often referred to as a
voodoo technique, crystallization conditions are tested in a high throughput method using 96-well screening plates, and any hits are further
optimized using a larger volume of the particular solution. While a crystallization condition may eventually be found, the process can take anywhere
from a few days to even a year or two to happen, making the crystallization process the rate-limiting step for protein crystallographers. During this
time, the protein must stay in solution and maintain its structure so as to produce a high quality crystal; a condition that is not often the case.
Similarly, a stable, highly concentrated protein sample is required to perform many of the more advanced NMR experiments. This is because many
of these experiments require days and even weeks to run, during which the homogeneity of the solution is key to acquiring quality spectra. Should
the protein unfold or precipitate out of solution during an experiment, the resulting chemical change would either not produce any signal, or one
which could not be used for structure/dynamics determination.
For Cryo-EM, working with frozen-hydrated specimens brings a number of challenges both for manipulations and imaging. When handling cryo-EM
grids to load them into the microscope, exposure to atmospheric water vapour rapidly leads to frost buildup on the grid. Under the TEM, these ice
crystals on the grid surface appear as huge boulders that completely block the electron beam. Thus, grids are kept under liquid nitrogen as much as
possible to minimize frost contamination. Problems with ice conditions are common—insufficient rapid freezing leads to formation of hexagonal
ice, while devitrification occurs when samples warm up, leading to formation of cubic ice. Various degrees of contamination may occur, and frosting
at atmospheric pressure causes the above-mentioned ice crystal deposition, while contamination within the column or under low-vacuum
conditions gives rise to a more subtle artefact.
One of the hallmarks of protein crystallography is that size does not matter. Whether one is working with a 25 kDa monomeric protein, or a 900
kDa multimeric complex, if it can be crystallized and produce a high-resolution diffraction pattern its structure can be determined. This is due to the
fact that once in crystal form, a protein is in a more-or-less static conformation which, after passing it through the x-ray beam at different angles,
can produce a single structural model. Cryo-EM is similar in this regard. Very large structures, including massive nucleoprotein complexes, such as
the ribosome, can be elucidated using Cryo-EM. The same cannot be said for NMR.
In NMR, the protein is in a soluble state and therefore in constant movement. The most important movement that governs the spectral quality is
that of the molecular tumbling rate. For proteins larger than about 40 kDa, the tumbling rate decreases significantly, in turn increasing the
transverse relaxation rate (T2). Essentially, this results in a weaker and rapidly decaying NMR signal, which manifests itself in peak broadening and
spectral overlap.
One of the major advantages of NMR is its ability to record small and large-scale protein dynamics, a phenomenon that is generally suppressed
when a protein is crystallized. Although a crystallized protein may exhibit a certain amount of motion within the lattice, the motions manifest
themselves as static or dynamic disorder, the former of which may result in two different conformations of a particular region, and the latter in
averaged electron density. In general, crystallization may restrict a protein’s natural flexibility and motions. Cryo-EM suffers from this same
limitation as samples are frozen and immobile. However, cryo-EM is capable of capturing a snap shot of the native structure as freezing is
instantaneous and does not require the formation of a crystal lattice.
Crystallography, however, is not left in the cold when it comes to dynamic structure analysis. Time-resolved crystallography can be used to monitor
changes in the protein structure upon addition of some ligand, or change in the environment. Because all protein crystals are highly hydrated, they
are able to serve as crucibles for some biochemical reactions. The crystal is typically soaked in a solution containing the ligand of interest to initiate
the biochemical reaction, after which the crystal is quickly placed into the beam-line and diffraction pattern is obtained. This can be performed
multiple times if necessary to obtain a variety of structural intermediates. The process though requires many things to go right: the protein cannot
become disordered nor should the crystal become cracked during the soaking process, and a high-powered synchrotron isrequired to collect high-
quality diffraction data over short exposure times.
In the end, protein x-ray crystallography, cryo-EM and NMR spectroscopy are not mutually exclusive techniques; one can easily pick up where the
other falls short. In analyzing NMR dynamics experiments, for example, one can greatly benefit from existing crystal structure data, or cryo-EM data
onto which the NMR structural data can be superimposed. Similarly, NMR structure data can be used to supplement a cryo-EM or crystal structure
with more information on the protein’s dynamics, binding information, and conformational changes in solution.
back to the top

The proteome is the entire set of proteins that is produced or modified by an organism or system. Proteomics has enabled the identification of ever
increasing numbers of protein. This varies with time and distinct requirements, or stresses, that a cell or organism undergoes. Proteomics is an
interdisciplinary domain that has benefitted greatly from the genetic information of various genome projects, including the Human Genome
Project. It covers the exploration of proteomes from the overall level of protein composition, structure, and activity. It is an important component
of functional genomics.
After genomics and transcriptomics, proteomics is the next step in the study of biological systems. It is more complicated than genomics because an
organism’s genome is more or less constant, whereas proteomes differ from cell to cell and from time to time. Distinct genes are expressed in
different cell types, which means that even the basic set of proteins that are produced in a cell needs to be identified.
In the past this phenomenon was assessed by RNA analysis, but it was found to lack correlation with protein content. Now it is known that mRNA is
not always translated into protein, and the amount of protein produced for a given amount of mRNA depends on the gene it is transcribed from
and on the current physiological state of the cell. Proteomics confirms the presence of the protein and provides a direct measure of the quantity
present.
A cell may make different sets of proteins at different times or under different conditions, for example during development, cellular differentiation,
cell cycle, or carcinogenesis. Further increasing proteome complexity, as mentioned, most proteins are able to undergo a wide range of post-
translational modifications.
Therefore, a proteomics study may become complex very quickly, even if the topic of study is restricted. In more ambitious settings, such as when a
biomarker for a specific cancer subtype is sought, the proteomics scientist might elect to study multiple blood serum samples from multiple cancer
patients to minimise confounding factors and account for experimental noise. Furthermore, many proteins undergo postranslational modifications
such as phosphoyrlation. Many of these post-translational modifications are critical to the protein’s function. Thus, complicated experimental
designs are sometimes necessary to account for the dynamic complexity of the proteome.
3.6 References

3.6 References
Molnar, C. and Gair, J. (2013) Antibodies. Chapter in Concepts in Biology, Published by B.C. Open Textbook Project. Available at:
https://opentextbc.ca/biology/chapter/23-3-antibodies/
The Human Atlas Project. (2019) Methods. Available at: https://www.proteinatlas.org/learn/method
Uhlén M et al, 2015. Tissue-based map of the human proteome. Science
PubMed:25613900 DOI:10.1126/science.1260419
Thul PJ et al, 2017. A subcellular map of the human proteome. Science.
PubMed:28495876 DOI: 10.1126/science.aal3321
Uhlen M et al, 2017. A pathology atlas of the human cancer transcriptome. Science.
PubMed:28818916 DOI:10.1126/science.aan2507
Ahern, K. and Rajagopal, I. (2019) Biochemistry Free and Easy. Published by Libretexts. Available at:
https://bio.libretexts.org/Bookshelves/Biochemistry/Book%3A_Biochemistry_Free_and_Easy_(Ahern_and_Rajagopal)/09%3A_Techniques/9.04%
3A_Gel_Exclusion_Chromatography.
Magdeldin, S. (2012) Gel Electrophoresis – Principles and Basics. Published by InTech under Creative Commons Attribution 3.0. Available at:
https://pdfs.semanticscholar.org/4b93/70ac3946cec6e12c369679c4178a5ef38e61.pdf
Structural Biochemistry/Proteins/X-ray Crystallography. (2018, November 19). Wikibooks, The Free Textbook Project. Retrieved 15:40, August 17,
2019 from https://en.wikibooks.org/w/index.php?title=Structural_Biochemistry/Proteins/X-ray_Crystallography&oldid=3488057.
UCD: Biophysics 200A (2019) “NMR Spectroscopy vs X-ray Crystallography”, Chapter published in Current Techniques in Biophysics. Published by
Libretexts and available at: https://phys.libretexts.org/Courses/University_of_California_Davis/UCD%3A_Biophysics_
200A_-_Current_Techniques_in_Biophysics/NMR_Spectroscopy_vs._X-ray_Crystallography
Wikipedia contributors. (2019, June 27). Protein purification. In Wikipedia, The Free Encyclopedia. Retrieved 23:32, July 28, 2019, from
https://en.wikipedia.org/w/index.php?title=Protein_purification&oldid=903657925
Wikipedia contributors. (2019, February 15). Fast protein liquid chromatography. In Wikipedia, The Free Encyclopedia. Retrieved 17:14, August 15,
2019, from https://en.wikipedia.org/w/index.php?title=Fast_protein_liquid_chromatography&oldid=883530035
Wikipedia contributors. (2019, July 9). Protein mass spectrometry. In Wikipedia, The Free Encyclopedia. Retrieved 15:27, August 16, 2019, from
https://en.wikipedia.org/w/index.php?title=Protein_mass_spectrometry&oldid=905547289
Wikipedia contributors. (2019, July 8). Peptide synthesis. In Wikipedia, The Free Encyclopedia. Retrieved 06:13, August 17, 2019, from
https://en.wikipedia.org/w/index.php?title=Peptide_synthesis&oldid=905401648
Wikipedia contributors. (2019, July 11). Edman degradation. In Wikipedia, The Free Encyclopedia. Retrieved 06:16, August 17, 2019, from
https://en.wikipedia.org/w/index.php?title=Edman_degradation&oldid=905808326
Bhella, D. (2019) Cryo-electron microscopy: an introduction to the technique, and considerations when working to establish a national facility.
Biochysical Reviews 11: 515-519. Available at: https://link.springer.com/article/10.1007/s12551-019-00571-w

Electrophoretic mobility
Monday, 18 October, 2021 02:34 PM
Gel Electrophoresis is a technique used to separate DNA, RNA or protein molecules based on their
size, electrical charge and conformation using electric field at a definite pH.
It is based on the principle that charged molecules when placed in an electric field, tend to migrate
towards the electrode of the opposite charge.
Many important biological molecules, such as amino acids, peptides, proteins, nucleotides and
nucleic acids, possess ionisable groups and, therefore, at any given pH, exist in solution as electrically
charged species, either cations (positively charged) or anions (negatively charged).
Gel Electrophoresis makes use of porous gels that act as a molecular sieve separating bigger
molecules from the smaller ones.
The rate at which each molecule travels through the gel is called its electrophoretic mobility and its
determined mainly by its net charge, size & shape.
• Strongly charged molecules move faster than weakly charged ones.
• Smaller molecules move faster than larger ones.
• Molecules with highly coiled structures move faster than uncoiled ones.
Nature of charge:
• Under the influence of an electric field these charged particles will migrate either to cathode
or anode depending on the nature of their net charge.
• Nucleic acids which are negatively charged, due to the presence of the sugar-phosphate
backbone, migrate toward the anode which is positively charged.
• Amino acids that make up proteins may be positive, negative, neutral, or polar in nature
depending on the functional groups present in the amino acid. The charges of amino acids
together give a protein its overall charge.
• Thus, when researchers want to separate proteins using gel electrophoresis, they must first
mix the proteins with a detergent called sodium dodecyl sulfate (SDS). This treatment makes
the proteins unfold into a linear shape and coats them with a negative charge, which allows
them to migrate toward the positive end of the gel and be separated.
Voltage:
When a potential difference (voltage) is applied across the electrodes, it generates a potential
gradient (E), which is the applied voltage (V) divided by the distance “d” between the two electrodes
i.e.
E = V/d
When this potential gradient ‘E’ is applied, the force as the molecule bearing a charge of ‘Q’ is
F=ExQ
Unit of Force (F) is Newtons & Unit of Charge (Q) is coulombs
It is this force that drives the molecule towards the electrodes.
Frictional force:
There is also a frictional force that retards the movement of this charged molecule.
• This frictional force depends on :
• Hydrodynamic size of the molecule
• Shape of the molecule
• Pore size of the medium in which the electrophoresis is taking place
• Viscosity of the buffer.
The velocity ‘v’ of the charged molecule is an electric field is therefore given by the equation :
V = (E x Q)/f =(V x Q)/(d x f)
where ‘f’= frictional coefficient
Electrophoretic mobility:
Electrophoretic mobility ( u ) of an ion is represented as the ratio of the velocity of the ion and the
field strength. i.e.
u =V/E
When a p.d. is applied, the molecule with different overall charges will begin to separate owing to
their different electrophoretic mobility.

their different electrophoretic mobility.
Even the molecule with similar charges will begin to separate if they have different molecular
sizes, since they will experience different frictional forces.
Current:
According to Ohm’s law:
V/I=R
Therefore, it appears that it is possible to accelerate an electrophoretic separation by increasing the
applied voltage, which ultimately results in corresponding increase in the current flowing.
The distance migrated by the ions will be proportional to both current and time.
Heat:
One of the major problems for most forms of electrophoresis, that is the generation of heat.
During electrophoresis, the power ( W ) generated in one supporting medium is given by
W= I2R
Most of the power generated is dissipated as heat.
The following effects are seen on heating of the electrophoretic medium has:
• An increased rate of diffusion of sample and buffer ions which leads to the broadening of the
separated samples.
• Formation of convention currents, which leads to mixing of separated samples.
• Thermal instability of samples that are sensitive to heat.
• A decrease of buffer viscosity and hence reduction in the resistance of the medium.
If a constant voltage is applied, the current increases during electrophoresis owing to the decrease in
resistance and this rise in current increases the heat output still further.
For these reasons, often a stabilized power supply is used, which provides constant power and thus
eliminates fluctuations in heating.
Constant heat generation is however a problem. For which the electrophoresis is run at very low
power (low current) to overcome any heating problems, but this can lead to poor separation as a
result of the increased amount of diffusion due to long separation time.
Compromise condition have to be found out with reasonable power settings, to give acceptable
separation time and an appropriate cooling system, to remove liberated heat. While such system
works fairly well, the effect of heating are not always totally eliminated.
Electroendosmosis:
Electroendosmosis occurs due to the presence of charged groups on the surface of the support
medium.
For instance, paper has some carboxyl group present, agarose contains sulfate groups depending on
the purity grade and the surface of glass walls used in capillary electrophoresis contains silanol (Si-
OH) groups.
These groups, at appropriate pH, will ionize, generating charged sites.
It is these charges that generate electroendosmosis.
In case of capillary electrophoresis, the ionized sianol groups creates an electrical double layer, or a
region of charge separation, at the capillary wall/electrolytic interface.
When voltage is applied cations in the electrolyte near the capillary walls migrate towards the
cathode, pulling electrolyte solution with them.
This creates a net electro osmotic flow towards cathode.
From <https://fromthelazybiologist.wordpress.com/2021/05/27/gel-electrophoresis/>

MOLECULAR METHODS 1
Monday, 18 October, 2021 02:55 PM
MOLECULAR METHODS 1

Protein Purification & Separation
Chromatography
• The source of a protein is generally tissue or microbial cells. The first step in any protein purification procedure is to break open these cells,
releasing their proteins into a solution called a crude extract. If necessary, differential centrifugation can be used to prepare subcellular
fractions or to isolate specific organelles.
• Commonly, the extract is subjected to treatments that separate the proteins into different fractions based on a property such as size or
charge, a process referred to as fractionation. Early fractionation steps in a purification utilize differences in protein solubility, which is a
complex function of pH, temperature, salt concentration, and other factors.
• The solubility of proteins is lowered in the presence of some salts, an effect called 'salting out'. The addition of certain salts in the right
amount can selectively precipitate some proteins, while others remain in solution. Ammonium sulfate ((NH4)2SO4) is particularly effective and is
often used to salt out proteins. The proteins thus precipitated are removed from those remaining in solution by low-speed centrifugation.
• Dialysis is a procedure that separates proteins from small solutes by taking advantage of the proteins’ larger size. The partially purified
extract is placed in a bag or tube made of a semipermeable membrane. When this is suspended in a much larger volume of buffered solution of
appropriate ionic strength, the membrane allows the exchange of salt and buffer but not proteins. Thus dialysis retains large proteins within the
membranous bag or tube while allowing the concentration of other solutes in the protein preparation to change until they come into equilibrium
with the solution outside the membrane. Dialysis might be used, for example, to remove ammonium sulfate from the protein preparation.
• Column Chromatography: A porous solid material with appropriate chemical properties (the stationary phase) is held in a column, and a buffered
solution (the mobile phase) migrates through it. The protein, dissolved in the same buffered solution that was used to establish the mobile
phase, is layered on the top of the column and allowed to percolate into the solid matrix by the addition of the buffer solution on top. The
protein solution forms a band within the mobile phase that is initially the depth of the protein solution applied to the column. As proteins
migrate through the column, they are retarded to different degrees by their different interactions with the matrix material. Individual types
of proteins gradually separate from each other, forming bands within the broader protein band.
• Separation improves (i.e., resolution increases) as the length of the column increases. However, each individual protein band also broadens with
time due to diffusional spreading, a process that decreases resolution.
• Three types of column chromatographic methods used in protein purification.

○ (a) Ion-exchange chromatography exploits differences in the sign and magnitude of the net electric charges of proteins at a given pH.
○ (b) Size-exclusion chromatography, also called gel filtration, separates proteins according to size.
○ (c) Affinity chromatography separates proteins by their binding specificities.
• Ion-Exchange Chromatography: The column matrix is a synthetic polymer (resin) containing bound charged groups; those with bound anionic
groups are called cation exchangers, and those with bound cationic groups are called anion exchangers. The affinity of each protein for the
charged groups on the column is affected by the pH (which determines the ionization state of the molecule) and the concentration of competing
free salt ions in the surrounding solution. Separation can be optimized by gradually changing the pH and/or salt concentration of the mobile
phase so as to create a pH or salt gradient. In cation-exchange chromatography, the solid matrix has negatively charged groups. In the mobile
phase, proteins with a net positive charge migrate through the matrix more slowly than those with a net negative charge, because the migration
of the former is retarded more by interaction with the stationary phase.

of the former is retarded more by interaction with the stationary phase.
• Size-Exclusion Chromatography: In this method, large proteins emerge from the column sooner than small ones. The solid phase consists of
cross-linked polymer beads with engineered pores or cavities of a particular size. Large proteins cannot enter the cavities and so take a shorter
(and more rapid) path through the column, around the beads. Small proteins enter the cavities and are slowed by their more labyrinthine path
through the column. Size-exclusion chromatography can also be used to approximate the size of a protein being purified similar to gel
electrophoresis.
• Affinity Chromatography: The beads in the column have a covalently attached chemical group called a ligand—a group or molecule that binds to
a macromolecule such as a protein. When a protein mixture is added to the column, any protein with affinity for this ligand binds to the beads,
and its migration through the matrix is retarded. For example, if the biological function of a protein involves binding to ATP, then attaching a
molecule that resembles ATP to the beads in the column creates an affinity matrix that can help purify the protein. As the protein solution
moves through the column, ATP-binding proteins (including the protein of interest) bind to the matrix. After proteins that do not bind are
washed through the column, the bound protein is eluted by a solution containing either a high concentration of salt or free ligand—in this case,
ATP or an analog of ATP. Salt weakens the binding of the protein to the immobilized ligand, interfering with ionic interactions. Free ligand
competes with the ligand attached to the beads, releasing the protein from the matrix; the protein product that elutes from the column is often
bound to the ligand used to elute it.

bound to the ligand used to elute it.
• Chromatographic methods are typically enhanced by the use of HPLC, or high-performance liquid chromatography. HPLC makes use of high-
pressure pumps that speed the movement of the protein molecules down the column, as well as higher-quality chromatographic materials that can
withstand the crushing force of the pressurized flow. By reducing the transit time on the column, HPLC can limit diffusional spreading of protein
bands and thus greatly improve resolution.
Electrophoresis
The process of the separation of proteins is based on the migration of charged proteins in an electric field is called electrophoresis.
Disadvantages Advantages
• Generally not used to purify proteins, because simpler alternatives • Generally used for protein analysis
are usually available • Proteins can be visualized as well as separated, permitting a researcher to
• Electrophoretic methods often adversely affect the structure and estimate quickly the number of different proteins in a mixture or the degree
thus the function of proteins of purity of a particular protein preparation.
• Electrophoresis can be used to determine crucial properties of a protein such
as its isoelectric point and approximate molecular weight

Screen clipping taken: 18-10-2021 02:53 PM
• Electrophoresis separates proteins on the basis of mass or charge. SDS gel electrophoresis and isoelectric focusing can be used separately or in
combination for higher resolution.
• All purification procedures require a method for quantifying or assaying the protein of interest in the presence of other proteins. Purification
can be monitored by assaying specific activity

Csir-Ugc Net

Uploaded by

Copyright:

Available Formats

Csir-Ugc Net

Uploaded by

Document Information

Original Description:

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Csir-Ugc Net

Uploaded by

Copyright:

Available Formats

Amino Acids

• Building blocks of polypeptides/proteins

Structural Features of Amino Acids

• Each sulfur-containing aa can link to other sulfur-containing aa through

Stereoisomers of Amino Acids

• D- and L- notation provides a quick shorthand for designating enantiomers.

Amino Acids Can be Classified by R Group

Absorption of light by molecules - Lambert-Beer Law

Amino Acids Can Act as Acids and Bases

What information do we get from titration curves?

Peptides Are Chains of Amino Acids

Peptides Can Be Distinguished by Their Ionization Behavior

Some Proteins Contain Chemical Groups Other Than Amino Acids

Amino Acid Sequencing - Edman Degradation

Amino Acid Sequencing - Mass Spectrometry

What Biological Functions Do Proteins Perform?

Chapter 3: Investigating Proteins – Chemistry

3.1 Protein Purification

3.1 Protein Purification

Protein purification is either preparative or analytical.

Precipitation and Differential Solubilization

Figure 3.3 Sucrose Density Gradient.

Hydrophobic Interaction Chromatography (HIC)

Ion Exchange Chromatography

3.2 Protein Identification and Visualization

Detection of Proteins in Gels

Two-Dimensional Gel Electrophoresis

Antibody Structure and Production

Enzyme-Linked Immunosorbent Assay (ELISA) and Microarrays

Figure 3.24. Different Reporter Systems.

Protein Sequencing using Edman Degradation

3.4 Protein Structure Elucidation

Nuclear Magnetic Resonance (NMR) Imaging

Advantages and Disadvantages to Structure Elucidation

3.5 Proteome Analysis

• A molecule with n chiral centres has 2n different possible stereoisomers.

For note making:::

1. MOLECULES AND THEIR INTERACTION RELAVENT TO BIOLOGY

A) Membrane structure and function

4. Cell communication and cell signaling

6. SYSTEM PHYSIOLOGY - PLANT

7. SYSTEM PHYSIOLOGY - ANIMAL

A) Mendelian principles : Dominance, segregation, independent assortment.

9. DIVERSITY OF LIFE FORMS:

A. Principles & methods of taxonomy:

10. ECOLOGICAL PRINCIPLES

11. EVOLUTION AND BEHAVIOUR

A. Emergence of evolutionary thoughts

12. APPLIED BIOLOGY:

13. METHODS IN BIOLOGY

B. Histochemical and Immunotechniques

H. Methods in field biology:

At present, the biological classification includes:

Animal Classification Page 62

Animal Classification Page 63

Animal Classification Page 64

Viruses, Viroids and Lichens

Animal Classification Page 66