Electrophoresis 1

ELECTROPHORESIS
• Separation of charged molecules in a solution based on their tendency to
move towards electrodes, by applying an electric field is called
electrophoresis.

• Developed by Tiselvis and Longsworth in 1937.

• Biomolecules such as nucleotides, amino acids, RNA , DNA and proteins are
charged molecules. So , they can be separated by means of electrophoresis.

PRINCIPLE

• If a direct current is passed through a solution containing charged

molecules, the positively charged cations (+) will migrate towards cathode
(-) and the negatively charged anions (-) will move towards the anode (+).

• The mobility of the molecules, however , depends on the net charge, size,
and shape of the molecule, pH, composition of the suspending medium,
and the applied current between the electrodes.
 Electrophoresis 2

• A supporting medium or matrix is required to separate molecules according

to their size and charge. It should be an inert, non- toxic material with
specific porosity.

• It should have a good filtering effect to separate molecules based on their

size. It should not interfere with electrical conductivity of buffer. Paper
strips, cellulose acetate, starch, agarose, polyacrylamide etc. are used as
supporting medium in electrophoresis.

• The mobility ( velocity) of molecules can be represented by the following

equation :

V = Eq/ ʄ

Where, v is the velocity of the migration of molecules,

E is the electric current ( in volts/cm),

q is the fractional co-efficient that accelerates the molecules to move

through the medium,

ʄ is the fractional resistance of the molecule.

ʄ is represented by the equation

ʄ = 6πηr

where, r is the radius of the molecule

η is the viscosity of the medium

• DNA and RNA are negatively charged molecules so that they migrate
towards the anode.

• The small molecules move faster through the supporting medium than the
large molecule. This principle is used in the separation of DNA and RNA.
 Electrophoresis 3

• The proteins also migrate through the supporting medium based on their
net electric charge at specific pH and size.

• The pH change affects the movement of molecules through the pores of

the supporting medium.in order to keep the medium at a constant pH,
buffers are used to maintain pH.

• Barbitone buffer, phosphate buffer, TBE buffer, TAE buffer, tris- glycine
buffer etc. are commonly used.

• An electrophoresis set- up consists of two buffer tanks, two platinum

electrodes, a supporting material (gel), buffer and sample.

• A stable DC of 5-8V/cm is suitable for the separation of charged molecules

by using electrophoresis.

• The rate of electrophoresis depends on the following factors:

▪ pH of the medium

▪ strength of electric field

▪ size of the molecules

▪ temperature

▪ concentration of sample.

APPLICATIONS

• separation of proteins from a mixture

• separation of RNA from a mixture

• used for the separation of DNA from a mixture

• used for the separation of immunoglobulins

• used for the separation of plasma protein

• used for the separation of lipoproteins

 Electrophoresis 4

• used for the determination of molecular weight of proteins and DNA

• used for the determination of size of DNA fragments

GEL ELECTROPHORESIS
• In gel electrophoresis, gels are used as the medium . when the gel is packed
as a column it is called column zone electrophoresis.

• If the gel is coated on glass plates, it is called open block electrophoresis.

• Disc electrophoresis is a column zone electrophoresis because gel is packed

as vertical cylindrical column in tubes.

POLYACRYLAMIDE GEL ELECTROPHORESIS ( PAGE )

Separation of molecules based on their charges and size using polyacrylamide gel
as a separating medium is called PAGE.

PRINCIPLE

• If a direct current is passed through a polyacrylamide gel containing

charged molecules, the cations (+) will migrate towards cathode (-) and the
anions (-) will move towards the anode (+).

• Proteins and nucleic acids are negatively charged molecules, they tend to
move towards the anode.

• The small molecules move faster through the supporting medium than the
large molecule.

• Mobility of the molecules depends on the net charge, size, shape, pH,
composition of the suspending medium and current.

• Polymerization of acrylamide in the presence of methylene bisacrylamide

and ammonium persulphate, gives polyacrylamide gel.
 Electrophoresis 5

• When the gel is allowed to polymerize in small tubes sealed at the bottom
with a rubber cap and a layer of water on the top, it forms a column of
cross- linked matrix with uniform pores.

• Water at the top layer excludes air that interferes with polymerization.

• The pore size can be changed by altering the concentration of acrylamide in

the solution. 7.5 % acrylamide is used for the separation of proteins.

APPARATUS

• The PAGE apparatus consists of two buffer tanks and a set of glass tubes
between them.

• One buffer tank is called lower tank. It is a small glass chamber with a lid.
The lid has holes. Each hole is fixed with a glass tube using a rubber cork.

• The other buffer thank is known as upper tank. It bears holes at its bottom.
The upper end of glass tubes are fixed in each hole using a rubber cork.

• The glass tube is small tube with the size of 0.5- 1 cm diameter and 1cm
length.

• Gel column is prepared in the tube before assembling it in between the

tanks.

• The buffer thanks are filled with buffer.

• One platinum electrode ( cathode) is inserted into the upper tank and
another one (anode) is placed in the lower tank.
 Electrophoresis 6

WORKING

• Rubber corks are placed over the bottom of hollow glass tubes.

• Acrylamide solution containing 7.5% acrylamide, ammonium persulphate

and 1.1 ml of small pore solution (separation gel) , are filled in the glass
tube.

• 0.1ml of distilled water is added on the top of the acrylamide solution and
kept as such for 25-40 minutes for polymerization.

• The distilled water is removed and 0.2ml of 2.5% acrylamide solution

containing a large pore solution (spacer gel) is added to the top of the
solidified gel followed by 0.1 ml of distilled water and kept as such for 15
minutes for polymerization.

• Then the water layer is removed and 0.05 ml of sample is added followed
by 0.001 ml of marker dye.

• Eg: for the separation of albumin protein Bromophenol blue is used as dye
to indicate the progress of electrophoresis.

• The remaining space is filled with Tris buffer.

 Electrophoresis 7

• After removing the rubber corks, the rubber tubes are attached to the
compartments in such a way that one end of a tube is attached to the
anode compartment and the other end to the cathode compartment.

• Then the compartments are filled with 200 ml of Tris buffer and current is
applied.

• When the marker dye migrates to the required position in the tube, the
electrophoretic run is stopped and the tubes are removed from the
compartments.

• The gels are immediately removed from the tubes with the help of a
syringe needle and each gel is stored in 70% acetic acid contained in a test
tube.

• The gels are stained with appropriate dyes and sample is quantified
spectrophotometrically or densitometrically.

• If the sample contains proteins, the rods are stained with amidoblack or
coomassie blue.

• After washing the gel with acetic acid, the colour bands are visualized for
proteins.

• If the sample contains DNAs, the gel rods are stained with ethidium
bromide and visualized under a UV transilluminator for light bands.

APPLICATIONS

• PAGE is used for the separation and purification of DNAs and proteins in
biological samples.

• It is used to separate proteins based on charge, but not mass.

• It is used to determine the molecular weight of proteins and DNAs.

• It is also employed for quantifying proteins and DNAs.

 Electrophoresis 8

• It is used for PCR

• It is used for DNA sequencing.

Types of PAGE

1. SDS PAGE

2. NON SDS PAGE

1.SODIUM DODECYL SULPHATE- POLYACRYLAMIDE GEL

ELECTROPHORESIS OR SDS- PAGE
• SDS-PAGE (Polyacrylamide Gel Electrophoresis), is an analytical method
used to separate components of a protein mixture based on their size
using polyacrylamide gel as a separating medium and sodium dodecyl
sulphate (SDS) as a detergent to neutralize the charge of proteins.

PRINCIPLE

• The technique is based upon the principle that a charged molecule will
migrate in an electric field towards an electrode with opposite sign.

• SDS is an anionic detergent.

• It unfolds proteins.

• Different protein molecules with different shapes and sizes, needs to be

denatured done with the aid of SDS

• SDS binds to the non covalent bonds of protein molecules so that the
proteins lose their secondary, tertiary or quaternary structure and
unfolded into extended polypeptide chains ( linear structure).

• SDS is anionic ,it binds to all positive charges on a protein, effectively

coating the protein in negative charge.
 Electrophoresis 9

• The proteins being covered by SDS are negatively charged and when loaded
onto a gel and placed in an electric field, it will migrate towards the anode
(positively charged electrode) are separated by a molecular sieving effect
based on size.

• After the visualization by a staining technique, the size of a protein can be

calculated by comparing its migration distance with that of a known
molecular weight ladder (marker).

INSTRUMENTATION AND WORKING

1.Sample preparation

• Samples may be any material containing proteins .

• The sample to analyze is optionally mixed with chemical

denaturant SDS ,bromophenol blue (Dye) , glycerol and βmercaptoethanol.

• Heating the samples to at least 60-95 °C promotes denaturation.

• SDS is an anionic detergent that denatures secondary and tertiary

structures, and additionally applies a negative charge to each protein in
proportion to its mass.
 Electrophoresis 10

2.Preparation of polyacrylamide gel

• The separating and stacking gels typically consist

of acrylamide, bisacrylamide, denaturant (SDS), ammonium persulphate
and a buffer with an adjusted pH .

• pH for separating gel is 8.8 and pH for stacking gel is 6.8

• The acrylamide concentration of the gel can also be varied, generally in the
range from 5% to 25%.

• Lower percentage gels are better for resolving very high molecular weight
molecules, while much higher percentages of acrylamide are needed to
resolve smaller proteins.

• Gels are usually polymerized between two glass plates in a gel caster.

• The separating gel is prepared and poured into the space between the two
glass plates. Some amount of water is poured on the top of the gel is left as
such for 30-60 minutes for polymerization.

• Stacking gel is prepared and water is removed from the separating gel and
stacking gel is poured on the separating gel.
 Electrophoresis 11

• Gels are usually polymerized between two glass plates in a gel caster, with
a comb inserted at the top to create the sample wells.

• After the gel is polymerized the comb can be removed and the gel is ready
for electrophoresis.

3.Electrophoresis

• Various buffer systems are used in PAGE depending on the nature of the
sample and the experimental objective.

• The buffers used at the anode and cathode may be the same or different.

• Gel plate is placed between the electrodes. Positive electrode positioned at

the bottom of the gel and negative electrode positioned at the top of the
gel.

• The gel is inserted into a chamber and tris-glycine- chloride buffer with a pH
8.3 poured into the tank.

• SDS is present in both the gel and the running buffer to ensure the
denaturation of protein.

• During sample application in addition to samples a molecular weight size

marker of known sizes are also added along with the samples. This helps to
 Electrophoresis 12

determine the approximate molecular weight of sample proteins by

comparing the distances .

• Each sample is added to separate wells and the samples contain

bromophenol blue and glycerol.

• Bromophenol blue act as a dye help in visualization of sample and glycerol

increases the density of the sample.

• An electric field is applied across the gel, causing the negatively charged
proteins or nucleic acids to migrate across the gel away from the negative
and towards the positive electrode (the anode).

• Depending on their size, each biomolecule moves differently through the

gel matrix.

• The stacking gel ensure that all the proteins in the sample enters the
separating gel at the same time.

• The running buffer consist of glycine and chloride ions .

• Stacking gel has a pH of 6.8 so, glycine carries neutral charge as a result
they move very slowly as compared to chloride and sample protein.

• As a result protein is trapped between glycine and chloride ions at stacking

gel.

• Then the protein , glycine and chloride ions enter into the separating gel
with a pH 8.8 and glycine became negative and migrate more fast as
compared to chloride and protein.

• Smaller proteins migrate in fast rate as compared to larger ones .

• Due to the small size bromophenol blue migrate fast in gels.

• After separation, glass is removed and the stacking gel is discarded.

 Electrophoresis 13

4.Detection

• Following electrophoresis, the gel may be stained ,for proteins, most

commonly with Coomassie Brilliant Blue in the presence of acetic acid
.After staining different proteins appear as distinct bands within the gel.

• Western blotting

• Autoradiography allowing visualization of the separated proteins.

APPLICATIONS

• Measuring molecular weight of protein.

• Peptide mapping.

• Estimation of protein size.

• Determination of protein subunits or aggregation structures.

• Estimation of protein purity.

• Protein quantitation.

• Monitoring protein integrity.

• Comparison of the polypeptide composition of different samples.

• Analysis of the number and size of polypeptide subunits.

 Electrophoresis 14

• Post-electrophoresis applications, such as Western blotting.

• Detection of Protein Ubiquitination.

2.NON SDS
• Gel electrophoresis run in the absence of SDS is called NON SDS or "Native"
or "non-denaturing" gel electrophoresis.

• It is a type of PAGE Used to analyze proteins that are still in their folded
state.

• It uses no charged dye so the electrophoretic mobility of proteins is related

to the intrinsic charge of the proteins.

• The migration distance depends on the protein charge (depend on amino

acid), its size and the pore size of the gel.

• This was the original mode of electrophoresis.

PRINCIPLE

• NON SDS Polyacrylamide gel electrophoresis (Native Page) uses a non –

denaturing gel.

• Therefore, SDS or any other denaturing agent is not added to the gel
matrix. In Native Page, the separation of proteins is based on the charge
and the size of the protein. Therefore, the mobility of the protein depends
on the charge and the size of the protein.

• The charge of the protein depends on the side chains of the amino acids.

• If the side chains are negatively charged, the protein will receive an overall
negative charge and vice versa.

• Proteins retain a 3D conformation due to the folding that takes place.

 Electrophoresis 15

• Folding results from the several bond types in proteins such as disulfide
bonds, hydrophobic interactions and Hydrogen bonds.

• Therefore, if the NON SDS PAGE is carried at a neutral pH, the proteins will
be separated according to the molecular shape of the protein. Therefore,
NON SDS PAGE can be used as a sensitive technique to detect the change
in charge or conformation of the protein.

• The main advantage of native PAGE is that the protein used for the PAGE
analysis can be recovered in its original state after the PAGE analysis, as the
protein is not disturbed during the process, and the stability of the protein
is increased.

• Use polyacrylamide gel as the matrix of the gel.

• Can be done in a vertical manner or horizontal manner.

• The electrophoresis apparatus including the gel tank, tubes, the power
supply are required.

• The visualizing of the gel can be done by staining methods

WORKING

• The apparatus consists of a buffer reservoir system and a power pack.

• The buffer reservoir system has an upper cathode compartment and lower
anode compartment containing buffer solution.

• According to the purpose same or different buffers are used to fill anode
and cathode compartments.

• Glass tubes having 1.4 cm length and 0.5 cm diameter connect the two
compartments.

• The compartment is provided with platinum electrodes.

• One end of the tube is tightly closed with a rubber cork and filled with 1.1
ml of small pore solution ( separation gel ).
 Electrophoresis 16

• Above this solution , 0.1 ml of double distilled water is added to polymerize

for 30 minutes.

• After the removal of water layer, 0.2 ml of large pore solution ( spacer gel )
is overlaid followed by 0.1 ml of distilled water for polymerization ( 15
minutes).

• Then the water layer is removed and 0.005 ml of sample is added followed
by 0.001 ml of marker dye.

• The remaining space of the tube is filled with Tris buffer.

• After removing the rubber corks, the tubes are attached to the
compartments in such a way that one end of the tube is attached to the
anode compartment and the other end to the cathode compartment.

• Then the compartments are filled with tris buffer (200 ml) and current is
applied.

• When the marker dye migrates to the required position in the tube, the
electrophoretic run is stopped and tubes are removed from the
compartments.

• The gels are immediately removed from the tubes with the help of a
syringe and each gel is stored in 70% acetic acid contained in the test tube.

• The gels are stained with appropriate dyes and the sample is quantified
spectrophotometrically or densitometrically.
Electrophoresis 17
 Electrophoresis 18

APPLICATIONS

• Used to detect the change in charge or conformation of the protein.

• Used to detect the amino acid content of protein

• Used to detect protein- protein interaction

• Used to detect nucleotide binding regions of proteins

• Used to detect structures like molten globule or some motifs of protein

• Separation of acidic proteins

• Identification of proteins present in Bovine Serum Albumin (BSA).

TWO DIAMENSIONAL GEL ELECTROPHORESIS (2 D GEL

ELECTROPHORESIS)
• 2D gel electrophoresis (2DE) is a key technique for purifying individual
proteins from complex samples based on their isoelectric points and
molecular weights.

PRINCIPLE

• It is a powerful tool and is designed by combining the resolving power of

isoelectric focusing with SDS- PAGE.

• As the molecular weight and isoelectric point of a macromolecule are not

related with each other, this makes use of these two properties of molecule
with great resolution power.

• First dimension: Separation according to proteins isoelectric points (pI)

• Second dimension: Separation according to molecular weight by SDS PAGE

 Electrophoresis 19

WORKING

• Isoelectric focusing can be combined with SDS-PAGE to obtain very high

resolution separations.

• A single sample is first subjected to isoelectric focusing on gel in a capillary

tube.

• In isoelectric focusing , a pH gradient is maintained with gradual increase

from anode to cathode .

• When the protein sample is introduced into the system has a net positive
charge and will migrate towards the cathode.

• Thus at a particular pH (isoelectric point ) , the net charge of the protein

becomes zero and will not move further.

• The pH at which the ionic charge of a solution is equal to the ionic charge of
a protein is called isoelectric point of the protein.

• At this point, the gel is removed from capillary tube.

• This single-lane gel is then placed horizontally on top of an SDS-

polyacrylamide slab.

• They then undergo electrophoresis to yield a two-dimensional pattern of

spots.

• In such a gel, proteins have been separated in the horizontal direction on

the basis of isoelectric point and in the vertical direction on the basis of
mass.

• After fractionation a specific protein can be identified by western blotting ,

fluorescent probes etc.

• Or identify proteins by coupling two-dimensional gel electrophoresis with

mass spectrometric techniques.

• By staining methods (Coomassie Brilliant Blue, silver stain, copper stain).

 Electrophoresis 20

APPLICATION

• 2-D electrophoresis applications include protein expression analysis,

• Biomarker analysis

• Discovering new drug targets

• Detecting post- or co-translational modifications.

• Studying protein expression in normal, disease, or developmental states

• Identifying novel proteins.

 Electrophoresis 21

ISOELECTRIC FOCUSING (IEF)

• IEF, also known simply as electrofocusing, is a technique for separating
charged molecules, usually proteins or peptides, on the basis of their
isoelectric point (pI).

• The isoelectric point (pI) is the pH value at which the molecule carries no
electrical charge ( Neutal).

• In this method, electric current forces the protein molecules to reach the
isoelectric point in the pH gradient.

• There is no further movement of proteins after reaching the same pH as

their isoelectric point.

• The IEF is very useful for the separation of closely related proteins having
the same size and weight but different isoelectric points.

PRINCIPLE

• Proteins are amphoteric in nature as amino acids are amphoteric.

• An amphoteric molecule can either act as an acid or base based on the

surrounding medium.

• According to the pH of the medium charge of the protein will differ.

• Proteins contain many positively and negatively charged groups on their

surface. Therefore, each protein has some net ionic charge.

• Solutions also have some ionic charge depending on the cations and anions
in them.

• In basic environment – the acidic group of protein became negatively

charged.

• In acidic environment – the basic group of protein became positively

charged.
 Electrophoresis 22

• In isoelectric point – protein have zero charge.

• Proteins exist as cations (+) at pH values below the isoelectric point. They
exist as anion (-) at pH range above the isoelectric point.

• At the isoelectric point, the number of negative charge on the protein is

equal to the number of positive ions in the solution. So, the net charge is
zero.

• Since the electrical potential difference is zero, proteins do not moves

further while moving across the pH gradient. Here, proteins are focused on
their isoelectric pH of the medium.

• Many closely related proteins differ in their isoelectric pH because of the

presence of different acidic and basic groups. Hence, such proteins can be
separated by IEF.
 Electrophoresis 23

WORKING

• The apparatus consists of a lower buffer tank, an upper buffer tank and a
glass tube. The glass tube is fixed in between the two tanks. One electrode
is fixed in each buffer tank.

• A short glass tube with open ends is taken and cleaned well.

• The lower end of the tube is closed with a cork.

• A sucrose density gradient column with pH gradient is prepared in the tube.

• Ampholyte containing 1.94g acrylamide, 0.06g bisacrylamide, 5g sucrose,

0.25ml riboflavin solution and 40ml of water is prepared. The ampholytic
solution is poured slowly along the wall of the tube until it reaches the level
of the column inside.

• The tube is kept undisturbed for 3hrs for the polymerization of

polyacrylamide gel with a pH gradient.

• After polymerization of the gel, the cork at the base is removed.

• A sample containing proteins with bromophenol blue dye is poured on the

top of the pH(high pH) gradient column. Most of them are negatively
charged at this pH.

• The tube is attached with two buffer reservoirs.

• A solution with more acidic pH is filled in one buffer reservoir and another
solution with more alkaline pH is filled in the other buffer reservoir.

• Electrodes are connected and the electrophoresis unit is switched on.

• As the protein move through the pH gradient they gain positive charge and
reach neutrality.

• After reaching the isoelectric pH, there is no further movement of the

protein. This movement can be seen by the movement of bromophenol
blue dye added to the protein solution.
 Electrophoresis 24

• After disconnection, the particular band is taken from the gel to isolate the
protein.
 Electrophoresis 25

APPLICATIONS

• The IEF is very useful for the isolation and purification of proteins having
the same mass but different isoelectric points.

• It is a sensitive technique to resolve closely related proteins.

• For research in cytology, taxonomy and immunology

• Used for the separation and identification of serum proteins.

• Identification of isoenzymes

• Used by food & agricultural industries, forensic and human genetic

laboratories.

DENSITY GRADIENT GEL ELECTROPHORESIS

• This is again a polyacrylamide gel system, but instead of running a slab gel
of uniform pore size throughout (15% gel) a gradient gel is formed, where
the acrylamide concentration varies uniformly from typical 5% at the top of
the gel to 25% acrylamide at the bottom of the gel.

• The higher the polyacrylamide concentration, the smaller the pore sizes in
the matrix. Higher concentration gels can separate or resolve smaller sized
proteins, while lower concentration gels, with larger pore sizes, are better
at resolving higher molecular weight proteins.

• Used to separate proteins according to their Molecular weight/mass.

PRINCIPLE

• Like fixed concentration polyacrylamide gels, gradient gels rely upon the
“sieving” effect created by the matrix of polymerized acrylamide.

• Higher the polyacrylamide concentration, the smaller the pore sizes in the
matrix.
 Electrophoresis 26

• Higher concentration gels can separate or resolve smaller sized proteins,

while lower concentration gels, with larger pore sizes, are better at
resolving higher molecular weight proteins.

• Unlike fixed concentration gels, gradient gels are formulated with a range
of polyacrylamide concentrations, where the continuous gradient begins
with a lower concentration and ends with a higher concentration.

• Gradient gels are normally run as SDS gels with a staking gel.

• There are two advantages to running gradient gels. First, a much greater
range of protein Mr values can be separated than on a fixed-percentage gel.

• In a complex mixture, very low molecular weight proteins travel freely

through the gel to begin with, and start to resolve when they reach the
smaller pore sizes towards the lower part of the gel.

• Much larger proteins, on the other hand, can still enter the gel but start to
separate immediately due to the sieving effect of the gel.

• The second advantage of the gradient gels is that proteins with very similar
Mr values may be resolved, although they cannot otherwise be resolved in
fixed percentage gels.

• As each protein moves through the gel the pore sizes become smaller until
the protein reaches its pore size limit.

• The pore size in the gel is now too small to allow passage of the protein,
and the protein sample stacks up at this point as a sharp band.

• A similar-sized protein, but with slightly lower Mr, will be able to travel a
little further through the gel before reaching its pore size limit, at which
point it will form a sharp band.

• These two proteins, of slightly different Mr values, therefore, separate as

two close sharp bands.
 Electrophoresis 27

INSTRUMENTATION

• Unlike fixed concentration gels, gradient gels are formulated with a range
of polyacrylamide concentrations, where the continuous gradient begins
with a lower concentration and ends with a higher concentration.

• Glass chambers: uses two chambers, where one chamber contains the
acrylamide solution at the lowest gradient concentration, and the other
contains the higher acrylamide concentration.

• Acrylamide solutions: The higher percentage acrylamide (e.g., 25%) is

poured between the glass plates first and a continuous gradient of
decreasing acrylamide concentration follows.

• Gradient mixer :The gradient is formed via a gradient mixer and run down
between the glass plates of a slab gel.

• Peristaltic Pump : Peristaltic pump is used to move the acrylamide solution

from the lower and higher concentration chamber into the gel casting
plates.

• Gel casting plates.

 Electrophoresis 28

• Comb : a comb inserted at the top of the gel to create the sample wells.

• Platinum cathode and anode.

• Bufffer.

• Buffer reservoir system and a power pack.

WORKING

• The gradient is formed via a gradient mixer and run down between the
glass plates of a slab gel.

• The higher percentage acrylamide (e.g., 25%) is poured between the glass
plates first and a continuous gradient of decreasing acrylamide
concentration follows.

• Acrylamide concentration varies uniformly from typical 5% at the top of

the gel to 25% acrylamide at the bottom of the gel.

• Gels are usually polymerized between two glass plates in a gel caster.

• When polymerized, pour the stacking solution .

• Insert a comb and wait until polymerized.

• After the gel is polymerized the comb can be removed and the gel is ready
for electrophoresis.

• Sample is added to separate wells.

• Gel plate is placed between the electrodes that are placed at the two
buffer tanks and filled with buffer.

• Apparatus is turn on and the electrophoretic run is carried out and

separation take place .
 Electrophoresis 29

• At the top of the gel there is a large pore size (5% acrylamide) but as the
sample moves down the gel the acrylamide concentration slowly increases
(25%) and the pore size correspondingly decreases.

• Sample proteins are able to travel a little further through the gel before
reaching its pore size limit, at which point it will form a sharp band.

• The gels are stained with appropriate dyes and sample is quantified
spectrophotometrically.
 Electrophoresis 30

APPLICATIONS

• More range of proteins can be separated.

• Used to Separate proteins with very similar Mr values can be separated.

DISC ELECTROPHORESIS
• Discontinuous electrophoresis (disc electrophoresis) is a type
of polyacrylamide gel electrophoresis.

• It was developed by Ornstein and Davis.

• This method produces high resolution and good band definition.

• It is widely used technique for separating proteins according to size and

charge.

PRINCIPLE

• In this method, the gel is divided into two parts, separating and stacking
gel, both have different concentrations of polyacrylamide and pH.

• The one with lower concentration is stacked on top of the one with higher
concentration.

• Discontinuity is based on four parameters:

➢ Gel structure,

➢ pH value of the buffer,

➢ Ionic strength of the buffer, and

➢ The nature of the ions in the gel and electrode buffer.

 Electrophoresis 31

INSTRUMENTATION AND WORKING

• Consists of two buffer systems, electrodes, tube.

• The tube is separated into two parts having different porosity and buffered
at different pH.

• The macromolecular mixture migrates from the more porous gel into less
porous gel accompanied by a change in pH.

• As a result each protein becomes concentrated as a very thin, sharp band

producing much higher resolution than can be achieved in a continuous
buffer.

• The gel is preferred for disc electrophoresis is polyacrylamide. There are

two different porosity gels – stacking gel ( high porosity) and running or
separating ( low porosity ) are used.

STACKING GEL

• The upper stacking gel is prepared by using about 2-3% acrylamide and is
highly porous and devoid of any molecular sieving action.

• The buffer used in this gel is usually Tris- Hcl with a pH 6.7

SEPARATING GEL

• The lower separating gel is prepared by using about 5-10% acrylamide

and pores are numerous and of a smaller diameter imparting molecular
sieving property to this gel.

• The buffer used in this gel is usually Tris- Hcl with a pH 8.9

• In this gel macromolecules are subsequently separated.

• Cathode and anode buffer system is filled with Tris- Glycine –Chloride
buffer with a pH 8.3
 Electrophoresis 32

• Sample with marker dye and Tris –Hcl buffer with a pH of 6.7 is injected
into the stacking gel and the power is turned on.

• Separation take place according to the sample size and charge.

• Glycine has neutral charge at pH 6.7 of stacking gel, so it has low mobility.
As a result the protein is trapped between glycine and chloride ions at
stacking gel.

• Then the protein , glycine and chloride ions enter into the separating gel
with a pH 8.9 and glycine became negative and migrate more fast as
compared to chloride and protein and protein is easily separated.

• Smaller proteins migrate in fast rate as compared to larger ones .

• After separation the gel may be stained ,for proteins, most commonly
with Coomassie Brilliant Blue in the presence of acetic acid

• After staining different proteins appear as distinct bands within the gel.

• Western blotting or Autoradiography also used for the visualization of the

separated proteins.
Electrophoresis 33
 Electrophoresis 34

APPLICATION

• It is widely used technique for separating proteins according to size and

charge.

• Separation of human serum proteins.

• Estimation of protein size , shape, conformation

• Estimation of protein purity.

HIGH VOLTAGE ELECTROPHORESIS (HVE)

• Also called High Voltage Paper Electrophoresis

( supporting medium is filter paper).

• As the name describe, the electrophoresis is carried under very high

voltage.

• The voltage applied was ranging from 2500-10000V or 500 mA.

• This is required for the substances of lower molecular weight which will
have considerable high diffusion rate like amino acids, peptides etc.

• These resulted in better resolution, better yield and very rapid separations
(10-60 minutes).

PRINCIPLE

• The basic principle of HVE, a form of zone electrophoresis, is that the

sample is dried on a sheet of paper, which is then wetted with an aqueous
buffer and subjected to a voltage gradient.

• The compounds in the sample migrate as zones towards the anode or

cathode according to their net charge.

• The higher the voltage applied, the faster the compounds migrate; fast
migration minimises diffusion of the spots.
 Electrophoresis 35

• However, an excessively high voltage would overheat the wet paper and
possibly degrade the compounds of interest.

• A cooling system must therefore also be used.

• HVE in one direction can be combined with chromatography in another,

which is at right angles to the first. This allows better resolution of
substances with similar charge pattern.

• Amino acids, peptides and amines have been successfully isolated by this
technique which yields results comparable to two dimensional
chromatography.

• It is possible to perform HVE in two directions at two different pH values.

• The sample is applied at the center of the long paper strip. The
electrophoretic run is carried out with two different pH one after the other.
Between the two runs the paper has to be dried. The paper also has to be
equilibrated two times with two different buffers corresponding to the pH
of each run. This method has yielded superlative resolution of amino acids
and peptides.

• The separated compounds can be detected either by staining,

autoradiography or fluorography, after which the sample can usually be
recovered for further work.

• Alternatively, the compounds can be eluted from a preparative paper ready

for further analysis, e.g. by MS, NMR or bioassay.

INSTRUMENTATION AND WORKING

HVPE are of two types

1.Immersed strip method

2.Flat-Bed System or Enclosed strip method

 Electrophoresis 36

1.IMMERSED STRIP METHOD

INSTRUMENTATION

• Apparatus consists of 42 × 57-cm Sheets of filter paper :

two types of paper are commonly used: Whatman No. 1 and the thicker
Whatman 3MM Chr.

• Glass tank & Glass trough: Glass trough is required to fill cathode buffer
and anode buffer is filled in glass tank.

• Glass rod for holding the wet paper in the glass trough.

• Aqueous running buffer: Formic acid/acetic acid/H2O, pH 2.0

or Acetic acid/pyridine/H2O, pH 3.5 are used.

• Platinum cathode and anode.

• Coolant : white spirit, toluene.

• Steel cooling coils in the top of the coolant to keep its temperature below
about 30°C.

• Lid.

WORKING

• Sample is applied to the filter paper and strip is immersed in large volume
of buffer and electrodes are placed at the two buffer tanks (Glass trough
and glass tank )and filled with buffer.

• Apparatus is turn on and the electrophoretic run is carried out and

separation take place.

• The compounds in the sample migrate as zones towards the anode or

cathode according to their net charge.
 Electrophoresis 37

• The higher the voltage applied, the faster the compounds migrate; fast
migration minimises diffusion of the spots.

2.FLAT-BED SYSTEM OR ENCLOSED STRIP METHOD

INSTRUMENTATION

• Apparatus consists of 30 × 57-cm sheets of filter paper.

• Polythene-insulated metal plate/ cooling plate / cooling sheet(containing

cooling coils).

• Insulated and padded lid/ pressure pad to maximise uniform contact with
the cooling plate.
 Electrophoresis 38

• Glass tanks: Two buffer tanks are required to fill the anode and cathode
buffer.

• Aqueous running buffer: Formic acid/acetic acid/H2O, pH 2.0 or Acetic

acid/pyridine/H2O, pH 3.5 are used.

• An insulated and padded lid is tightly clamped on top of the paper to

maximise uniform contact with the cooling plate.

• Insulated cover.

WORKING

• The paper is laid on a polythene insulated metal plate/sheet (containing

cooling coils) with the ends of the paper dipping into troughs containing
buffer and electrodes.

• Apparatus is turn on and the electrophoretic run is carried out and

separation take place. The compounds in the sample migrate as zones
towards the anode or cathode according to their net charge.

• The higher the voltage applied, the faster the compounds migrate; fast
migration minimises diffusion of the spots.
 Electrophoresis 39

DETECTION OF ANALYTES

• The separated compounds can be detected either by staining ,

autoradiography or fluorography, after which the sample can usually be
recovered for further work.

• Alternatively, the compounds can be eluted from the filter paper ready for
further analysis, e.g. by MS, NMR or bioassay.

APPLICATIONS

• Amino acids, peptides and amines have been successfully isolated by this
method.

• Detection of sugars, purine, pyrimidine’s and inorganic ions.

• The amino acid composition of two unknowns can be determined.

CAPILLARY GEL ELECTROPHORESIS

• CGE is a high-resolution and automated variation of the SDS-PAGE
technique used for the separation of Protein, DNA,RNA, Aminoacids.

• CGE is a subdivision of Capillary electrophoresis.

• In general, electrophoresis is a separation technique based on the

migration of charged molecules in response to an electric field, toward the
electrode of opposite charge. It is performed mainly in polyacrylamide gels.

• In CGE, the gel is located inside a capillary through which the sample
components must migrate. The larger the molecular weight, the longer the
migration time.

• So macromolecules will migrate according to their molecular mass.

 Electrophoresis 40

PRINCIPLE

• CGE uses separation based on the difference in solute size as the particles
migrate through the gel.

• Gels are useful because they minimize solute diffusion, prevent the
capillary walls from absorbing the solute, and limit the heat transfer by
slowing down the molecules.

• A commonly used gel apparatus for the separation of proteins is

capillary SDS-PAGE.

• It is a highly sensitive system and only requires a small amount of sample.

• In CGE the column is packed with a gel, which affects the motion of the
analytes.

• Accordingly, separation will be determined not only by the electrophoretic

force acting on the ions but also by the size of analyte molecules.

• A typical application is the separation of proteins in a capillary which is

filled with polyacrylamide gel and SDS.

• The presence of SDS aids the electrophoretic mobility of proteins, as it

coats their surface proportional to their size.

• Macromolecules will migrate according to their molecular mass. This

technique is very similar to SDS PAGE.

• A detection system, mostly UV or fluorescence detection, detects and

quantifies the components of the mixture.

• Molecular weight is determined in reference to a standard.

 Electrophoresis 41

Electrophoretic mobility ,µ
µ is the distance travelled by the particle in one second under a potential gradient
of one volt per cm.

µep = q6πŋr

where,

q = solute’s charge

ŋ = the buffer viscosity

r = solute’s radius.

Electrophoretic velocity , Vep is the solute’s velocity in a response to the applied

electric field.

Vep = µepE

µep = solute’s electrophoretic mobility

E = the magnitude of the applied electric field.

Vep and µep increases for more highly charged solutes and for solutes of smaller
size.

INSTRUMENTATION AND WORKING

• It consists of two platinum electrodes (anode and cathode) connected to a

high voltage power supply and a fused silica capillary tube filled with gel
matrix , whose ends are immersed into a reservoir containing buffer
solution.

• The sample is injected onto the capillary by temporarily replacing one of

the buffer reservoirs (normally at the anode) with a sample reservoir.
 Electrophoresis 42

• After replacing the buffer reservoir, an electric potential is applied across

the capillary and the separation is performed.

• As a result of electric field analytes migrate in the opposite charge

direction.

• Migration macromolecules through the porous gel is based on their charge

and mass and it is possible via an applied voltage.

• The smallest macromolecule migrates the fastest while the largest one
migrates to slowest.

• Samples and separations are detected by detectors and the connected to a

computer system to generate electropherogram.

• The analyte concentration is quantified more reliably through UV

absorbance, fluorescence, or mass spectrometry.
 Electrophoresis 43

APPLICATIONS

• Determination of molecular weight of protein, DNA & RNA

• Separation of protein, DNA, RNA, amino acids

• Protein characterization

• DNA sequencing

• Used in forensic science

• Drug analysing

EMSA OR ELECTROPHORETIC MOBILITY SHIFT ASSAY

• Interaction of protein with DNA is central to the control of many cellular
processes including DNA replication, recombination, repair, transcription,
translation, RNA processing , maturation, nuclear transport, formation of
cellular machinery etc.

• EMSA is a technique that is used to study the protein – nucleic acid

interaction.

• It is rapid, very sensitive technique.

• Also known as Bind shift assay and Gel retardation assay.

PRINCIPLE

• Relies on the fact that protein – DNA complexes migrate more slowly than
free DNA molecules when subjected to non – denaturing polyacrylamide or
agarose gel electrophoresis.

• Migration of DNA molecule during gel electrophoresis varies with the size –
smaller molecules migrates faster than the larger ones.

• A reduction in electrophoretic mobility shows that a complex is formed

between DNA and protein.
 Electrophoresis 44

• Rate of DNA migration is shifted or retarded when bound to a protein. So

the assay is also referred to as gel shift or gel retardation assay.

Shift is primarily due to :

➢ Increase in the molecular weight

➢ Adoption of charge

➢ Conditional change in the nucleic acid conformation.

• The effect of protein binding on the mobility of DNA is analysed by PAGE

followed by autoradiography.

• Protein – DNA complexes formed of linear DNA fragment s results in

characteristic retarded mobility in the gel.

• If a circular DNA used the protein – DNA complex actually migrate faster
than the free DNA.

• Selection of nucleic acid is very important in EMSA

• Short DNA templates are commonly uses.

• Binding of protein and DNA is depends on pH and salt concentration.

• It is very important to provide an environment that is very close to the

physiological conditions of that particular organisms.

• Buffers used- MOPS, bis and tris- glycine buffer and phosphate buffer.

• Additives are also used . Additives are small neutral solutes such as
glycerol, sucrose used to stabilize the protein – nucleic acid interactions.
 Electrophoresis 45

INSTRUMENTATION AND WORKING

1.Preparation of protein sample

i.Cell disruption

• Different biological materials require different cell disruption strategies.

Use chemical inhibitors and temperature control to minimize the activity of
protease and other enzymes that may modify the protein composition of
sample.

ii.Protein solubilisation

• It can be done by solutions containing detergents, reducing agents, salts

that are compatible with the electrophoretic techniques used.

iii.Contaminant removal and Desalting

• Remove interfering substances.

iv.Quantification

• Determine concentration of protein in a sample by protein assay.

 Electrophoresis 46

2. Synthesis and labelling of Nucleic Aid

• There is no need for labelling DNA if large quantities of DNA are available.

• Because the DNA bands can be visualize by using ethidium bromide

staining.

• If low concentration of DNA is available they should be labelling before

performing the experiment.

• Labelling methods :

➢ DNA is radiolabelled with P32

➢ Non-radioactive labels are biotin, fluorophores etc.

3.Binding reaction

• Protein sample, binding buffer , poly( dl-dc) an exogenous nucleic acid as

a competitor to prevent nonspecific interaction .

• Incubate this mixture for 20 minutes at 4 ⁰C. Labelled DNA probe is then
added and incubated at room temperature for additional 20 minutes.

4. Non- denaturing Gel electrophoresis

• After the binding reaction, next step is the separation of free nucleic acids
from the complexes formed by a non-denaturing gel electrophoresis. PAGE
or Agarose gel can be used .

• Steps includes : gel casting, sample loading ( free DNA and bound DNA
with dye) and electrophoresis.

• Both polyacrylamide and agarose can be used for EMSA but polyacrylamide
gels offer better electrophoretic resolution for protein – DNA and protein -
RNA complexes.

• Binding of proteins will reduce the mobility of DNA.

 Electrophoresis 47

• Different proteins may retard a fragment to different extends, depending

on their size and shape.

• If two proteins bind to same DNA, they will reduce its mobility even further.

• The free DNA fragment migrated rapidly to the bottom of the gel, while
those fragments bound to protein are retarded.

5.Detection

• Staining nucleic acid with ethidium bromide. Commonly used when large
quantities of DNA used.

• Autoradiography on X ray film (If less quantity of DNA is available , it should

be labelled with radioactive isotopes P 32 )

• Non-radioactive labels are biotin, fluorophores etc.

 Electrophoresis 48

APPLICATION

1.Used for quantifying interactions between protein and DNA

2. Determination of binding affinities

3.Determine the dissociation constant

• Kd ( Dissociation constant) is the equilibrium constant that measures the

tendency of a complex to dissociate its components.

• Kd is calculated as the concentration when 50% of receptor is occupies for a

ligand interaction.

• Here, the receptor is the DNA, hence Kd is gotten when 50% of DNA is
bounded by protein.

• For this, a fixed concentration of DNA is titrated with excess protein. Bound
and free DNA are separated using EMSA. Measure density of bands, Kd -
protein concentration when 50% of DNA is free.

4.Proving the presence and characterization of Transcription Factor

• This assay can be used to determine, if a particular transcription factor is

present within the nuclei of the cells or tissue of interest.

5. Gel shift assays are not limited to protein – DNA complexes it is also used to
study protein – RNA and protein – peptide interaction using the same
electrophoretic principle.
 Electrophoresis 49

ALTERNATIVES AND VARIANTS OF EMSA

1.Supershift assay

• DNA- protein complexes are identified by using antibodies.

• The formation of an antibody-protein-DNA complex further hinders the

movement of the complex within the gel resulting in a “supershift”.

2.Chromatin immunoprecipitation ( ChIP)

• It captures protein DNA interactions on a cross- linking agent ( eg.

Formaldehyde).

• Antibodies are used to specifically immune – precipitate a desired protein

of interest and quantitative PCR is used to measure the quantity of DNA
bound to that protein.

3.DNA Footprinting

• Method used for identification of the binding site recognized by specific

protein. It is based on the fact that interaction of a protein to a unique DNA
sequence shield that region of DNA from further modifications by DNAse or
some other chemicals.

...................................................................................................................................