Antibodies to GST fusions
cbookste at MEDICINE.BSD.UCHICAGO.EDU
Mon Jun 8 08:44:31 EST 1998
In answer to Rob's query:
"Can anyone tell me of any potential problems (ie from cross-reactivity with
endogenous GST) when raising antisera (in rabbits) against GST fusions. The
antisera is to be used on human samples, the GST in the fusion is
Schistosoma japonicum (from Pharmacia pGEX plasmid)."
We have made many GST protein fusions and cross-reactivity with
endogeneous GST was never a problem. This has been true for human, rat or
mouse tissue or cell lines on Westerns. Some of our serum antibody has been
good enough to give specific signal on IH, probably because the antibody
for the target protein was very high titer. Usually, however, we have
affinity purified for IH and IF. If you think endogenous GST is a problem,
it is easy to column purify your crude serum to remove all GST antibodies.
What was a problem was poor cross-reactivity to the target protein. This
may be due to poor antigenicity of the chosen fusion sequence or to folding
of the fusion protein which buried the antigenic sites on the native
protein. In which case, you try again with a new construct.
One other word of advice: Always check your purified fusion protein by
SDS-PAGE before injecting the bunny. Any breakdown or spurious bands means
you should go back and redo the prep. And if you freeze your fusion
protein, check it right before you use it to re-inoculate.
Crescence Bookstein, Ph.D.
Research Associate/Assistant Professor
Dept of Medicine-GI mc 6084
Univ. of Chicago
5841 S. Maryland Ave.
Chicago, IL 60637
cbookste at medicine.bsd.uchicago.edu
Phone: 773/702-2283 Fax: 773/702-2281
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