ELECTROPHORESIS

METHODOLOGY
 Equal amount of buffer in both compartment .
 Sample is layered on supporting medium.
 Supporting medium is connected to buffer by paper wicks.
 Electric current is passed by means of power pack.
 After electrophoretic run fixatives for 10 min.(acetone/methanol)
 Staining (amido black , commasie blue)
 De-staining (acetic acid) until gel backgrounge white.
 Again fixative for 10 min.
 Let it dry
 Densitometry.
 Supporting medium
A) Paper electrophoresis :
It is most common method used in clinical laboratories..
Mainly used for separation of prorein ,aminoacid and
nucleotides .
Low voltage : protein
High voltage : aminoacids,nucleotides
Advantage :
- relatively cheap , easy to use .

Disadvantage :
- long time consuming
- albumin tends to adsorb paper causing trailing
-undesirable endosmosis effect
• Applications
• In determination of purity and m.w. of
proteins.
• Diagnosis of hemoglobinopathies like sickle
cell anemia and thalassemias
• Diagnosis of dyslipoproteinemia
• Separating serum proteins for diagnostic
purpose such as col .mm .ns. Infection.
 Supporting medium
C) Agar or agarose Gel :

Agar is made up of two component :

a) Agarose :
b) Agaropectine : responsible for electroendosmosis

- Most commonly used

- Little endosmosis
- Easily scanned with densitometer
 Supporting medium
D) PAGE : Poly acrylamide gel electrophoresis

• Polyacrylamide have molecular sieving properties.

• So separation is very efficient on the basis of MW and
charge.

Advantage :
- Gel can be produced in variety of pore size.
- Diffusion is effective , sharp bands are seen .
- Electroendosmosis & adsorption effect is minimum.
- Used for separation for enzymes and isoenzymes .
 Supporting medium

E) SDS- PAGE : Poly acrylamide gel electrophoresis

• Protein is boiled for 1-2 min with denaturing agent sodium

dodecyl sulphite.
• Negative charge of SDS will cover all protein molecule.
• Then separation on the basis of MW. ct is minimum.
• In agar gel electrophoresis only 5 bands are separated while
in PAGE more than 20 bands separated .
 PFGE
PULSED FIELD GEL ELECTROPHORESIS
• Separation of DNA using altering the strength
and direction of electrical field between
elctrodes is called PFGE.
• Application :
• PFGE is used to separate human chromosome
with sharper bands and higher resolution.
 G. Immunoelectrophoresis:
(Rocket electrophoresis)
• Electrophoresis separation is followed by
antigen antibody reaction .
• Serum is fractionated into more than 40 bands .
• So it is much more sensitive and specific than
ordinary electrophoresis.