MCB 307

ELECTROPHORESIS

Electrophoresis is migration of charged particles or molecules in a

medium under the influence of an applied electric field.

The Rate of migration of charged molecules depends upon following

factors:

(a) The strength of electric field, size and shape.

(b) Relative hydrophobicity of the sample.

(c) Ionic strength and temperature of the buffer.

(d) Molecular size of the taken biomolecule.

(e) Net charge density of the taken bio molecule.

(f) Shape of the taken biomolecule.

In the process of electrophoresis large molecules have more difficulty in

moving through the supporting medium (i.e., gel) whereas the smaller
medium has more mobility through it.

Classification of Electrophoresis:

All modern electrophoretic apparatus have supporting media. A

supporting medium is a physical support through which the charged
molecules get separated. It has two primary functions-adsorption and
molecular sieving of the taken molecules which are intended to be
separated.
Some of the frequently used supporting media are starch, agar,
polyacrylamide and agarose. Depending upon the presence or absence of
supporting media the electrophoresis can be classified as free
electrophoresis and zone electrophoresis.

A. Free Electrophoresis:

In this type of electrophoresis, a free electrolyte is taken in place of

supporting media. Nowadays this type of electrophoresis has become out-
dated and mostly used in non-biological experiments.

B. Zone Electrophoresis:

This is the most prevalent electrophoretic technique. In this type of

electrophoresis the separation process is carried out on a stabilizing
media.

The zone electrophoresis is of following types;

(a) Paper electrophoresis

(b) Cellulose acetate electrophoresis

(c) Capillary electrophoresis

(d) Gel electrophoresis – which further includes Agarose gel

electrophoresis, SDS PAGE, PFGE and two-dimensional electrophoresis.

Gel Electrophoresis

Gel electrophoresis involves the use of gel as supporting media for

separation of DNA, RNA or proteins under the influence of electric
charge. It is usually performed for analytical purposes but may be used as
a preparative technique to partially purify molecules prior to use for other
methods such as mass spectrometry, PCR, cloning, DNA sequencing and
immuno-blotting.

This is the most commonly used electrophoresis in biotechnology

laboratories and is used for almost all types of experiments in RD

A typical gel electrophoresis apparatus is of two kinds:

(a) Vertical Gel Apparatus:

It is used for the separation of proteins in SDS-PAGE.

(b) Horizontal Gel Apparatus:

It is used for immune-electrophoresis, isoelectric focusing and

electrophoresis of DNA and RNA in the agarose gel.

Agarose Gel Electrophoresis

 Agarose gel electrophoresis is a method of gel electrophoresis used

in biochemistry, molecular biology, genetics, and clinical
chemistry to separate a mixed population of macromolecules such
as DNA , RNA or proteins in a matrix of agarose.
 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.
 They are the most popular medium for the separation of moderate
and large-sized nucleic acids and have a wide range of separation.
Principle of Agarose Gel Electrophoresis

 Gel electrophoresis separates DNA fragments by size in a solid

support medium such as an agarose gel. Sample (DNA) are
pipetted into the sample wells, followed by the application of
an electric current at the anodal, negative end which causes the
negatively-charged DNA to migrate (electrophorese) towards the
bottom (cathodal, positive) end.
 The rate of migration is proportional to size: smaller fragments
move more quickly, and wind up at the bottom of the gel.
 DNA is visualized by including in the gel an intercalating
dye, ethidium bromide.
 DNA fragments take up the dye as they migrate through the
gel. Illumination with ultraviolet light causes the intercalated dye
to fluoresce.
 The larger fragments fluoresce more intensely. Although each of
the fragments of a single class of molecule are present
in equimolar proportions, the smaller fragments include less mass
of DNA, take up less dye, and therefore fluoresce less intensely.
 A “ladder” set of DNA fragments of known size can be run
simultaneously and used to estimate the sizes of the other unknown
fragments.

Requirements/ Instrumentation of Agarose Gel Electrophoresis

The equipment and supplies necessary for conducting agarose gel

electrophoresis are relatively simple and include:

1. An electrophoresis chamber and power supply

2. Gel casting trays, which are available in a variety of sizes and
composed of UVtransparent plastic. The open ends of the trays are
closed with tape while the gel is being cast, then removed prior to
electrophoresis.
3. Sample combs, around which molten medium is poured to form
sample wells in the gel.
4. Electrophoresis buffer, usually Tris-acetate-EDTA (TAE) or Tris-
borate-EDTA (TBE).
5. Loading buffer, which contains something dense (e.g. glycerol) to
allow the sample to “fall” into the sample wells, and one or two
tracking dyes, which migrate in the gel and allow visual monitoring
or how far the electrophoresis has proceeded.
6. Staining: DNA molecules are easily visualized under an ultraviolet
lamp when electrphoresed in the presence of the extrinsic fluor
ethidium bromide. Alternatively, nucleic acids can be stained after
electrophoretic separation by soaking the gel in a solution of
ethidium bromide. When intercalated into doublestranded DNA,
fluorescence of this molecule increases greatly. It is also possible
to detect DNA with the extrinsic fluor 1-anilino 8-naphthalene
sulphonate.
7. Transilluminator (an ultraviolet light box), which is used to
visualize stained DNA in gels.
Steps Involved in Agarose Gel Electrophoresis

1. To prepare gel, agarose powder is mixed with electrophoresis

buffer to the desired concentration, and heated in a microwave
oven to melt it.

The concentration of Agarose Gel

 The concentration of agarose is referred to as a percentage of

agarose to volume of buffer (w/v), and agarose gels are normally in
the range of 0.2% to 3%.
 The percentage of agarose used depends on the size of fragments to
be resolved.
 The lower the concentration of agarose, the faster the DNA
fragments migrate.
 In general, if the aim is to separate large DNA fragments, a low
concentration of agarose should be used, and if the aim is to
separate small DNA fragments, a high concentration of agarose is
recommended.
 2. Ethidium bromide is added to the gel (final concentration 0.5
ug/ml) to facilitate visualization of DNA after electrophoresis.
3. After cooling the solution to about 60oC, it is poured into a casting
tray containing a sample comb and allowed to solidify at room
temperature.
4. After the gel has solidified, the comb is removed, taking care not to
rip the bottom of the wells.
5. The gel, still in plastic tray, is inserted horizontally into the
electrophoresis chamber and is covered with buffer.
6. Samples containing DNA mixed with loading buffer are then
pipetted into the sample wells, the lid and power leads are placed
on the apparatus, and a current is applied.
7. The current flow can be confirmed by observing bubbles coming
off the electrodes.
8. DNA will migrate towards the positive electrode, which is usually
colored red, in view of its negative charge.
9. The distance DNA has migrated in the gel can be judged by
visually monitoring migration of the tracking dyes like
bromophenol blue and xylene cyanol dyes.

Applications of Agarose Gel Electrophoresis

Agarose gel electrophoresis is a routinely used method for separating

proteins, DNA or RNA.

 Estimation of the size of DNA molecules

 Analysis of PCR products, e.g. in molecular genetic diagnosis or
genetic fingerprinting
 Separation of restricted genomic DNA prior to Southern analysis,
or of RNA prior to Northern analysis.
  The agarose gel electrophoresis is widely employed to estimate the
size of DNA fragments after digesting with restriction enzymes,
e.g. in restriction mapping of cloned DNA.
 Agarose gel electrophoresis is commonly used to resolve circular
DNA with different supercoiling topology, and to resolve
fragments that differ due to DNA synthesis.
 In addition to providing an excellent medium for fragment size
analyses, agarose gels allow purification of DNA fragments. Since
purification of DNA fragments size separated in an agarose gel is
necessary for a number molecular techniques such as cloning, it is
vital to be able to purify fragments of interest from the gel.

Advantages of Agarose Gel Electrophoresis

 For most applications, only a single-component agarose is needed

and no polymerization catalysts are required. Therefore, agarose
gels are simple and rapid to prepare.
 The gel is easily poured, does not denature the samples.
 The samples can also be recovered.

Disadvantages of Agarose Gel Electrophoresis

 Gels can melt during electrophoresis.

 The buffer can become exhausted.
 Different forms of genetic material may run in unpredictable
forms.
Spectroscopy
 The study of the interaction between matter and electromagnetic
radiation
 It is the term used to refer to the measurement of radiation intensity
as a function of wavelength.
 Often use to describe experimental spectroscopic methods.
 Four different spectroscopic techniques are commonly used and
this include
 1. Ultraviolet-visible spectroscopy
2. Infrared spectroscopy
3. Nuclear magnetic resonance spectroscopy (NMR)
4. Mass spectrometry.

The first three are known as absorption spectroscopy

 Spectral measurement devices are referred to as spectrometers,

spectrophotometers, spectrographs or spectral analyzers.

Spectrophotometers

 A spectrophotometer is an instrument that measures the amount of

light absorbed by a sample.
 Spectrophotometer techniques are mostly used to measure the
concentration of solutes in solution by measuring the amount of the
light that is absorbed by the solution in a cuvette placed in the
spectrophotometer.
 Scientist Arnold J. Beckman and his colleagues at the National
Technologies Laboratory (NTL) invented the Beckman DU
spectrophotometer in 1940.
Principle of Spectrophotometer

The spectrophotometer technique is to measure light intensity as a

function of wavelength. It does this by diffracting the light beam into a
spectrum of wavelengths, detecting the intensities with a charge-coupled
device, and displaying the results as a graph on the detector and then on
the display device.

1. A sample solution is placed inside the spectrophotometer.

2. A light source shines light toward the sample.
3. A device called a monochromator splits the light into each color,
or rather, individual wavelengths (just like a raindrop makes a
rainbow). An adjustable slit allows only one specific wavelength of
light through to the sample solution.
4. The wavelength of light hits the sample, which is held in a little
container called a cuvette. We need to be careful when handling
cuvettes; even a slight fingerprint can interfere with the results.
 5. Whatever light passes through the sample is read and displayed on
the output screen.

Instrumentation of Spectrophotometer

The essential components of spectrophotometer instrumentation include:

1. A table and cheap radiant energy source

 Materials which can be excited to high energy states by a high

voltage electric discharge (or) by electrical heating serve as
excellent radiant energy sources.

2. A monochromator, to break the polychromatic radiation into

component wavelength (or) bands of wavelengths.

 A monochromator resolves polychromatic radiation into its

individual wavelengths and isolates these wavelengths into very
narrow bands. It could be prism (or) grating.

PRISMS:

 A prism disperses polychromatic light from the source into its

constituent wavelengths by virtue of its ability to reflect different
wavelengths to a different extent
 Two types of Prisms are usually employed in commercial
instruments. Namely, 600 cornu quartz prism and 300 Littrow
Prism.

GRATINGS:
  Gratings are often used in the monochromators of
spectrophotometers operating ultraviolet, visible and infrared
regions.

3. Transport vessels (cuvettes), to hold the sample

 Samples to be studied in the ultraviolet (or) visible region are

usually glasses (or) solutions and are put in cells known as
“CUVETTES”.
 Cuvettes meant for the visible region are made up of either
ordinary glass (or) sometimes Quartz.

4. A Photosensitive detector and an associated readout system

 Most detectors depend on the photoelectric effect. The current is

then proportional to the light intensity and therefore a measure of
it.
 Radiation detectors generate electronic signals which are
proportional to the transmitter light.
 These signals need to be translated into a form that is easy to
interpret.
 This is accomplished by using amplifiers, Ammeters,
Potentiometers and Potentiometric recorders.

Measuring Absorbance

The amount of light that makes it through the substance is displayed on

the output screen and is called absorbance. In order for this number to
mean anything, we need a standard curve. A standard curve is
determined by recording the absorbance of known concentrations of a
material.
Applications of Spectrophotometer

Some of the major applications of spectrophotometers include the

following:

 Detection of concentration of substances

 Detection of impurities
 Structure elucidation of organic compounds
 Monitoring dissolved oxygen content in freshwater and marine
ecosystems
 Characterization of proteins
 Detection of functional groups
 Respiratory gas analysis in hospitals
 Molecular weight determination of compounds
 The visible and UV spectrophotometer may be used to identify
classes of compounds in both the pure state and in biological
preparations.