RE: immunizing peptides
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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