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IMMUNOELECTROPHORESIS
Objective
Principle
The immunoelectrophoresis used to characterize antibodies. This
technique is based on the principle of electrophoresis of antigen and
immunodiffusion of the antigens with a polyspecific antiserum to form
precipitin bands.
ELECTROPHORESIS
During electrophoresis, molecules placed in an electric field acquire a
charge and move towards appropriate electrode. The mobility of
molecule is dependent on a numbers of factors:
1.Size/shape of molecule
2.pH
3.Heat generated by ionic strength buffer
4.Charge particles
IMMUNODIFFUSION
•Antigen thus resolved by electrophoresis is subjected to
immunodiffusion wit antiserum added in a cut of the agarose gel.
•Immunodiffusion is due to the diffusion, density, gradient of antigen
and antibody which formed at the zone of equivalence, antigen
antibody complex precipitate to form an opaque arc shaped line in
gel.
•This precipitate line indicates the presence of antibody specific to
that antigen.
Material provided
Antiserum A has antibody against whole serum and hence contains a
mixture of antibodies against serum proteins. It will thus form more
than one precipitin line indicating the heterogeneity of antisera
Antiserum B has antibody against IgG. It contain single antibody, so
only one precipitin line can be formed.
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[PRACTICAL 4] MTEB2404
Procedure
1.10ml of 1.5% agarose (0.15g/10ml) was prepared in 1X
electrophoresis buffer by heating slowly till agarose dissolves
completely.
2.The end of the glass was marked. It will be towards
negative electrode during electrophoresis
3.The glass plate was placed on a horizontal surface. 10 ml of
agarose solution was pipette and spread onto the plate.
4.The glass plate was placed on the template holder provided in
ETS-2 and fixes the template. Punch a 3mm well with the gel
puncher, towards the negative end
5.Cut two roughs with the gel cutter provided, but do not
remove the gel from the trough.
6.12-15ul of antigen was added to the gel.
7.The glass plate was placed in the electrophoresis tank such that
the antigen well is at the cathode / negative electrode. 1X
electrophoresis buffer was poured such that it covers the gel.
8.The voltage was set to 50-100V and electrophoreses until the blue
dye travels 3-4cm from the wells. If electrophoresis beyond 3 hours,
heat can be generated and this is not good for the result of this
experiment.
9.The gel was removed from both the troughs and the plate was
kept at room temperature for 15 min. 250ul of antiserum-A was
added in one of the troughs and antiserum B in the other
10.The plate was placed in a moist chamber and diffusion was
allowed to occur at room temperature for overnight.
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[PRACTICAL 4] MTEB2404
Precipitation (IgG)
Antiserum A
Mixed antigen
Antiserum B
Discussion
Immunoelectrophoresis identifies
proteins based on their combined
electrophoretic and immunological
properties.
Conclusion:
From this experiment, the antiserum B formed a dense precipitin arc
against IgG antiserum, thus this indicates the heterogenicity of
antiserum to antigen.
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