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Transcript
INTRODUCTION
 Electrophoresis is a migration of a charged particle in a
medium under the influence of an applied electric
field.
 The usual purposes of carrying out electrophoretic
experiments are :
1. To determine the number, amount and mobility of
components in a given sample or to separate them.
2. To obtain information about the electrical double
layers surrounding the particles.
TYPES OF ELECTROPHORESIS
1.PAPER ELECTROPHORESIS
1. In this the sample is applied as a circular support
on a strip of whatman filter paper moistened
with the buffer solution.
2. The ends of the paper are
immersed in separate reservoirs containing buffer
and in which the electrodes are fitted.
3. Upon passing electric current, the ions in the sample
migrate towards oppositely charged electrodes.
This method is suitable for separation of low
molecular weight compounds such as amino acids
and nucleotides.
APPLICATIONS
1.This is single most critical procedure in electrophoresis
process.
2.Special devices are available for this purpose.
3.There is no hard and fast rule for the time of
application of the sample to the
electrophoretogram.this can be applied before the
paper has been equilibrated with the buffer or after
it.
2. STARCH GEL ELECTROPHORESIS
1. Starch gels are prepared by heating and
then cooling a solution of partially
hydrolyzed starch in an appropriate
buffer.
2. The Separation is accomplished
due to differences in charge as well as the
size of molecules because of molecular
sieving effect of the gel.
AGAROSE GEL ELECTROPHORESIS
1. Agarose gels are more porous and have a large
pore size and therefore used to fractionate large
macromolecules such as nucleic acids.
2. Agarose gels are cast by boiling agarose in
presence of a buffer then poured into a mold and
allowed to harden to form a matrix.
3. Higher the agarose larger is the pore size.
4. When the electric feild is applied across the gel,
negatively charged molecules move towards
oppositely charged electrodes on the basis of
their molecular size.
5. Smaller DNA molecules will travel faster as
compared to larger ones.
Commonly used buffers are tris acetate, tris borate or tris
phosphate at a concentration of 50mM over a pH range
of 7.5-8.0.
3. POLYACRYLAMIDE GEL ELECTROPHORESIS
1. The components used in the formation of this gel are known to be
neurotoxins.
2. The most commonly used components to synthesize the matrix are
acrylamide monomer, N, N’-methylenebisacrylamide, ammonium
persulphate and tetramethylenediamine(TEMED).
3. These free radicals can activate acryl amide monomer including them
to react with other acrylamide monomer forming long chains.
4.This chains become cross-linked if the reaction is carried out in the
presence of N-N’-methylenebisacrylamide.
5.TEMED acts as a catalyst of gel formation because of its ability to exist
in free radical 0form.
6.The whole mixture then irradiated with UV light in the presence of
oxygen.
Buffer System
1. Gel electrophoresis can be carried out in buffers
ranging from pH 3-10.
2. Ionic strength of the buffer system is generally kept
low(0.01-0.1M) to reduce heat production depending
upon the type of buffer systems used, polyacrylamide
gel electrophoresis may be of the following kinds:
(1) Native continuous polyacrylamide gel electrophoresis
(2) Native discontinous polyacrylamide gel
electrophoresis or native disc gel electrophoresis
(3) SDS- polyacrylamide gel electrophoresis (reducing)
(4) SDS- polyacrylamide gel electrophoresis (nonreducing)
(1) Native continuous polyacrylamide gel electrophoresis
In this same buffer ions are present throughout the
sample, gel and electrode vessel reservoirs at constant
pH such that the proteins have native conformation
and biological activity and hence it is known as native
continuous PAGE. In this system, protein sample is
loaded directly onto the gel, referred to as resolving
gel, in which separation the sample components
occurs.
(2) Disc gel electrophoresis
Disc gel electrophoresis is a modification of a
conventional zone electrophoresis which allows
the sample to enter the gel as a sharp band, thereby
helping further resolution.
The two different porosity gels are used
1. Stacking gel ( high porosity)
2. Separating or running gel (low porosity).
(3)SDS-PAGE(Non-reducing)
1. In this system proteins are dissociated into their sub units using ionic detergent such as
sodium lauryl sulfate.
2. The protein mixture is denatured by heating the sample at 100⁰C in the presence of excess
SDS.
3. Under these conditions most of polypeptides bind SDS in a constant weight ratio (1.4g
SDS per g polypeptide) .
4. The intrinsic charge of polypeptide are insignificant as compared to negative charges
provided by the bound detergent and these migrate in polyacrylamide gels according to
their size or molecular mass.
(4) SDS-PAGE(Reducing)
1.It is similar to SDS-PAGE described above except that in addition to SDS.
2. In this method protein sample is boiled in presence of excess SDS and βmercaptoethanol to denature proteins their individual polypeptide.
3. SDS polypeptide have net negative charge and migrate towards anode on the basis of
their size or molecular weight.
SDS PAGE
APPLICATIONS OF GEL ELECTROPHORESIS
This technique is used in molecular biology
1.Determination of DNA sequences
2.Southern and northern blotting
3.Restriction mapping of DNA
APPLICATIONS IN PROTEIN STUDY
1.Determination of molecular weight of proteins by
electrophrosis.
2.Determination of subunit stoichiometry.
OTHER TYPES OF ELECTROPHORESIS
1. ISOELECTRIC FOCUSING
1. It is an electrophoretic method for separation of proteins which differ in their isoelectric
points.
2. The proteins are made to migrate in pH gradient maintained by mixture of low
molecular weight ampholytes.
3.Proteins migrate depending upon their charge till they reach a region where the pH
corresponds to respective isoelectric point at which pH protein posseses no net charge
and hence get focused.
APPLICATIONS OF ISOELECTROFOCUSING
1.Elecrofocusing has been used for separation and
identification of serum proteins
2. It is used by food and agricutural industries.
3. It is used by forensic and human genetics
laboratories.
4. It is used for research in enzymology,
immunology, and membrane biochemistry etc.
2. IMMUNOELECTROPHORESIS
1. It is a agar/agarose gel electrophoresis which exploits the separation of
proteins on the basis of their charge to mass ratio and their antigencity .
2. Protein mixtures are first separated by electrophoresis on agarose gel and
then allowed to interact with specific antibody preparation.
3. Antibody diffuses through the gel and forms visible precipitate with
electrophoresed proteins for which the antibody has affinity.
4. The technique is used in the determination of antigen concentration. As a
antibody moves into gel, it encounters antibody and precipitates when
antigen-antibody concentration and equivalent resulting in the gel.
 APPLICATIONS
1.It is used to diagnose multiple myeloma, a disease affecting the bone
marrow.
2. It is used to identify certain proteiin molecules produced by the immune
system.
3. It is used to measure the Ig present.
3.TWO DIMENSIONAL GEL ELECTROPHORESIS
1. This powerful technique combines the resolving
power of isoelectric focusing with SDS gel
electrophoresis.
2. The mixture is first subjected to isoelectric focusing
in capillary tubes and are separated according to
their charge.
3.Then sample is subjected to SDS gel electrophoresis
which separates the proteins according to their
molecular weights.
4. CAPILLARY ELECTROPHORESIS
1. The technique that has very high resolving power
and speed.
2. CE involve electrophoresis of sample in small narrow
capillary tubes and high voltage from 10-15 KV is
applied.
This technique is used to separate biological material
including amino acids, proteins, nucleic acids as well
as drugs or even metals. Very small amount of
sample are required for analysis.
APPLICATIONS OF CAPILLARY ELECTROPHORESIS
1. It is used in the isolation of the correct optical isomer.
2. CE plays an importan role in ensuring a constant
product quality of therapeutic glycoproteins.
3. It is used in forensic area to detect performance
enhancing drugs.
PROTEIN (WESTERN) BLOTTING
The transfer of proteins from an acrylamide gel
onto a more stable and immobilzing support
such as nitrocellulose filter is called as
protein blotting or western blotting.
Blotting of proteins bands allow them to become more accessible
for detection and identification using a variety of methods such as
glyco proteins etc and also filters can be stored for much longer
period. The transfer of proteins from gels can be acheived by any
of the methods such as capillary action, application of vaccum or
electrophoretically.