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Types of Electrophoresis PDF
Types of Electrophoresis PDF
Operational procedure:-
Disadvantages:
1. Adsorption:
But adsorption is minimized by using buffer, more alkaline then isoelectric point
of sample.
2. Endo – osmosis:
3. Diffusion of small molecules:
It is overcome by applying high voltage (~ 20 V cm-1) thereby reducing the time
required for separation. In case of high voltage paper electrophoresis, the paper is
clamped between 2 cooled plates to carry off the heat generated by the high voltage
current flow.
Operational procedure:-
Advantages:-
1. Very little adsorption even with macromolecules. Therefore suitable supporting medium
for separation of radiolabelled substances, immuno-diffusion & immuno-electrophoresis.
2. Better resolution in shorter time compared to paper electrophoresis has less hydrophilic
than paper & less buffer is held by it.
3. Thin ground of strips may be rendered translucent (permitting light to pass through
partially) after separation & staining by treatment with clearing agents such as whitemor-
oil 120 which facilitates quantification.
Applications:-
1. Separation of compounds like amino acids, peptides, nucleotides, nucleic acids &
charged carbohydrates.
2. Particularly used for separation of blood proteins like glycoprotein, lipoprotein &
hemoglobin.
Gels are water insoluble, hydrophilic & semisolid colloids prepared shortly before use.
Molecular sieving property of semi–rigid gel helps to separate large ionic compounds such as
proteins which have similar charge properties but differ in size & shape.
Starch gel is prepared by heating & cooling partially hydrolyzed starch in an appropriate
buffer. This causes the branched chain of amylopectin component of starch to intervene & form
is semi–rigid gel.
Strong less porosity gel is prepared by less than 8–15% (w/v) starch.
Operational procedure:-
Applications:-
Gels are water insoluble, hydrophilic & semi–solid colloids prepared shortly before use.
Molecular sieving property of semi–rigid gel helps to separate large ionic compounds such as
proteins which have similar charge properties but differ in size & shape.
Agar is a cheap, nontoxic & chemically well defined. The powdery mixture of agrose
containing two galactose based polymer – agrose & agropectin. Agar is soluble on boiling in
aqueous buffer to perform a gel of about 380C.
Operational procedure:-
Advantages:-
Disadvantage:-
Applications:-
Analysis of
Serum protein,
Hb varients &
Isoenzyme pattern.
1. Medium saturation:
If the supporting medium is gel, the medium of saturation is a necessary because, the
gel is saturated with buffer at the time of gel preparation.
Sample is applied by using micropipette as a small spot or narrow greak on or along the
suitable origin.
For paper and cellulose acetate: If the individual components of a miximum have
opposite charges. They migrate in both directions. The before sample is near the middle of the
supporting medium.
If the components are either positive / negative charged samples,they are applied near
end, farthest away from electrode to which they are moving.
For gel: In gel electrophoresis, sample is introduced to get stabs usually (or) a strip of
filter paper into narrow stabs cut into the gel surface.
Sample concentration: In electrophoresis, the sample concentration will be within the range of
1 - 3 mg / ml.
After applying the sample, power switched on (at required Voltage) for a period
necessary for separation.
The electrophoresis is completed the power is off. And the supporting medium is
removed.
Paper cellulose acetate strips removed and dried and kept oven at 110.C. (unless
hermoliable compounds are present)
Gel stabs removed without any pressure. Cylindrical is removed by forcing water from
hypothermic syringe around the walls supporting glass tubes allowing the gets to be excluded
under gel pressure
After removal, gel is immersed in 7% acetic acid (which is used as fixative) to minimize
diffusion of compounds.
If sample absorb or fluoresce under uv and support medium not agents they can be
identified.
If separation compound shows uv absorption after treatment with agents they can be
identified.
Eg: Amino acid, Peptide, Protein, fluoresce on reacting with dansyl cholor
If on treatment with reagents if the biological molecules produce structural coloured compound
they can be analyzed.
Enzyme Detection:
Method:1
Detected by immobilizing the non - ionic substrates during gel preparation and the
enzyme activity detected after electrophoresis incubation in a more appropriate buffer.
STEP: 1 STEP: 2
Method: 2
Enzyme separated (electrophoresis) is kept in close cont. with the supporting medium
containing substrate. The enzyme activity is localised.
HORIZONTAL TYPE:
Electrophoresis equipment required for horizontal system basically consist of two items
*Power pack
* An electrophoresis
POWER PACK:
The power pack provides a stabilized direct current and it controls both voltage (0 -
500V) and current output (0 - 150 A)
Electrophoresis Unit:
-- Electrodes
--Buffer reservoir
--Supporting medium
--Transparent insulating cover
There are two buffer reservoirs and each buffer reservoir consist of two compartments
separate compartments are necessary, because any changes in pH occurring at electrodes does
not affect the buffer that is in contact with the supporting medium.
Support for electrophoretic medium (Supporting medium) may be paper, cellulose acetate
or gel. The contact between the supporting medium and buffer reservoir through 'wick'.
The supporting medium is cast on a glass / plastic sheet and placed on a cooling plate. (
an insulated surface through which cooling water is passed to conduct away generated heat.
VERTICAL TYPE:
Electrophoretic equipment required for vertical system basically consist of two items.
A power pack and
An elactrophoresis unit
POWER PACK:
The power pack provides a stabilized direct current and it controls both voltage (0-500V)
and current output.
ELECTROPHORESIS UNIT:
--- Electrodes
--- Buffer reservoir
--- Supporting medium
--- Transparent insulating chamber
Electrodes are made up of platinum or stainless
There are two buffer reservoirs; upper buffer reservoir and lower buffer reservoir.
The supporting medium is gel especially poly acryl amide gel. The gel is formed between
two glass plates that are clamped together but held apart by plastic spacers. The plastic comb
(well forming template) is placed in the gel solution and is removed after polymerization to
provide loading wells for samples.