Antibodies to SDS-PAGE purified proteins
nancid1016 at aol.com
Sat Nov 12 19:15:35 EST 1994
In article <Cx44nB.B6 at gpu.utcc.utoronto.ca>, japhale at gpu.utcc.utoronto.ca
(J. Aphale) writes:
If you want to use SDS-PAGE purified proteins for immunizations, use 5-10
ug per band. Stain the bands and cut them out. You can then mix them
with PBS and pass them repetedly through an 18G needle to break up the
gel. Inject this mixture as you normally would.
There are some things you should know about this. Acrylamide is a very
strong immunogen and you will get a lot of antibodies to the acrylamide.
Also, the antigens in the gel will be denatured and the resulting
antibodies will recognize the denatured form of the antigen, but not
necessarily the native form.
You may want to try transferring the gel to nitrocellulose, first. You
can use Ponceau S to stain the NC to visualize the bands. It will wash
out with PBS. The NC can be dissolved in DMSO and precipitated with
0.1M bicarbonate buffer, to give an injectible form. Antibodies generated
this way will see both native and denatured forms of the antigen.
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