RE: immunizing peptides

From:Dorit Zharhary

Nicole,

Performing competition/ inhibition test is more sensitive and may possibly 
give you a more definite specificity answer.
What you need to do is to preincubate the specific antibody at its working 
concentration with the peptide at 1-20 ug/ml. Preincubation should be at 
least 2 hours, and overnight is best. After this preincubation, just use 
the antibody in any application you desire and proceed regularly. This 
preincubation should eliminate the positive result which you obtain when 
applying antibody (without the peptide).
Negative control: incubate with a non-specific peptide at the same 
concentration. this should not eliminate positive result.

Good Luck,

Dorit

-----Original Message-----
From:	Nicole Cooper [SMTP:ncooper@mail.rah.sa.gov.au]
Sent:	Thu 10 January 2002 2:08
To:	Histonet@pathology.swmed.edu
Subject:	immunizing peptides

I have had some immunizing peptides synthesized to some commercially
available rabbit polyclonals that I have been using.  I was wondering 
whether
anyone out there had protocols that they use for  testing the specificity 
of
their antibody with the peptide.  I have started testing the peptides 
against
the corresponding antibody, but seem to be getting some strange binding
results especially with peptide and anti-rabbit secondary alone.
Any help greatly appreciated.
Nicole





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