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 migration of charged particles or molecules
DEFINITION under the influence of electric field.

to separate charged molecules,

use like DNA, RNA and proteins according to
their size.
 Charge – higher the charge greater the
electrophoretic mobility.

Size-bigger the molecule greater are the

FACTORS
AFFECTING frictional force
ELECTROPHORETIC larger particles have smaller electrophoretic
MOBILITY mobility
Shape – rounded contours elicit lesser frictional
and electrostatic retardation compared to sharp
contours.
Therefore globular protein move faster than fibrous
protein.
 It is a technique used for the separation of
GEL Deoxyribonucleic acid, Ribonucleic acid or protein
ELECTRO molecules according to their size and electrical
PHORESIS
charge using an electric current applied to a gel
matrix.

Agarose gel and

Types of Gel
Polyacrylamide gel.
 A highly purified uncharged polysaccharide
derived from agar.
Used to separate macromolecules such as
nucleic acids, large proteins and protein
AGAROSE
GEL
complexes.
It is prepared by dissolving 0.7% agarose in
boiling buffer solution and allowing it to cool to
40 °C.
It is fragile because of the formation of weak
hydrogen bonds and hydrophobic bonds
 a.Barbiturate buffer or Barbital Buffer : pH 8.6
b. Agarose : 0.7% agarose in buffer.(70mg agarose
in10 ml of buffer=0.7% gel )
MATERIAL c. Stanning solution : Coomassie brilliant blue in
REQUIRED acetic acid and D.W.
(2.5 mg dye powder mixed with 100 ml of solution
prepared by mixing of 45 ml of methanol, 10 ml of
glacial acetic acid and 45 ml of D.W).
c. De-staining solution : 7% glacial acetic acid
solution.
 PROCEDURE

Serum proteins are separated on agarose gel.

A few microliters of serum is required.

Agarose is heated in buffer till agarose is dissolved.

When the prepared gel temperature comes down to about 600C-
700C, about 1.5 ml is pipetted out and gently applied to one end of
a clean slide and allowed to cover entire slide evenly.

The two buffer compartments are filled with equal volume of

buffer.
The agarose plate is kept in position inside the chamber.
 PROCEDURE

Two filter paper (Whatman 3) strips having the same width as the agar plate
are placed on both end of the plate and are connected to the buffer
compartments.

A thin strip of coverslip is soaked with the serum sample (mixed with
tracking dye bromophenol blue) and place it carefully on the gel at one third
of the distance from the cathode end of the plate.

The chamber is closed and current is adjusted at 200V.

After 2 hrs run, the current is turn off.

The agar plate is removed & immersed in staining solution for 20 – 30

minutes. The plate is washed with destaining solution to clear the
background color. The slide Dried & various bands are observed.
 Clinical Significance

In normal serum five well characterized band namely: albumin, α1, α2, β and y-globulin
are seen. Albumin moves fastest towards anode and y-globulin has lowest mobility.
Direct scanning in densitometer quantitates the bands.
In normal electrophoretogram, the properties of various protein bands are as follows :
albumin-56%
α1 globulin-3%
α2 globulin-13.5%
β globulin-15.5%
y globulin -12%
Since the electrophoretic pattern of serum proteins in certain diseases varies markedly
from a normal pattern, it is of great diagnostic significance in several condition like
nephrosis, liver diseases and multiple myeloma etc.
In nephrosis, albumin level decreases and α2 globulin level increases.
In chronic liver disease albumin band is affected.
In multiple myeloma an abnormal M band appears between β globulin and y globulin.