Antigen for immunisation of
rabbits or guinea pigs may be purified by
preparative SDS-PAGE. Following electrophoresis and staining by aqueous
Coomassie the band with the antigen is excised from the gel, cut into
small gel pieces and homogenised in liquid nitrogen in a large mortar.
The homogeneous powder is suspended in Freud'sches Adjuvans and
injected into rabbits.
- Dissolve the antigen sample in Lämmli buffer. Boil for 5
min, Spin.
- Prepare a large PA gel: 1-2 mm spacer, 16x16 cm plates.
Assemble the
glass plates according to the Dobberstein procedure with silicon tubing
as sealing.
- Prepare 50 ml for the separating gel and 10 ml for the
stacking gel.
- Use a comb with only two slots: a small one for the MW
marker, the
remaining slots as a single slot for the sample (takes several ml)
- Run the gel at 60 mA for several hours with ventilation.
- carefully dissemble the glass plates
- stain in aqueous coomassie (0.5% Coomassie blue R-250 in
water) overnight, recycle the staining solution
- wash several times with water. The destaining takes some
time.
- Take a picture on the copy maschine.
- cut the band with the antigen with a blade. Compare to the
molecular weight marker.
- cut the gel slice with a blade into small bits and pieces
as much as possible.
- Transfer the gel material into a mortar filled with liquid
nitrogen.
- Homogenise the small gel pieces with a pistle. Add liquid
nitrogen from time to time.
- Homogenise until a fine powder is obtained. Bigger pieces
must not be present.
- Transfer the powder into a 50 ml Falcon tube.
- Take a small aliquot of the powder, boil in Lämmli and
analyse by SDS-PAGE to check whether you excised the correct protein.
- The frozen powder is ready for immunisation. The remaining
SDS appears to be tolerated by the injected animal.
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