[Histonet] use of blocking peptide to test antibody specificity

From:"Amy Wagelie"

Hi list-
Does anyone have experience with testing for antibody specificity with
a blocking peptide in the context of a rabbit anti-serum?? This
antibody is one we generated in our lab by immunizing w/ a human GST
fusion protein; the resultant anti-serum works beautifully for both
Western and IHC.  I now want to test for specificity (in IHC) by
blocking with the immunizing protein; i.e., incubating the primary
antibody w/ the protein for an hour (?) before staining.  I have read
that a 5 to 10-fold excess of peptide should be sufficient; I have
about 400mcg of the immunizing protein left, but my problem is that I
don't know how much specific anitbody is present in the anti-serum.
I'm guessing I'll have to just guess--but the protein was a challenge
to prepare and it's a pretty valuable commodity.  Any suggestions?
Amy Wagelie-Steffen, M.D.
UCSD Allergy / Immunology Fellow

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