RE: Peptide competition Assay

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To:"" <>, "'Carol Burden'" <>
Date:Tue, 11 May 1999 10:43:38 +0200
Content-Type:text/plain; charset="iso-8859-1"


The best way to do it is in 2 steps:
1. You need to work with the antibody at sub-saturating concentrations in order to see competition and confirm specificity. Therefore, titrate your antibody and choose a concentration that gives you about 90% staining intensity.
2. Pre-incubate  the antibody (at the above concentration ) with several concentrations of your specific peptide and a non-specific one. The latter is very important because peptides at high concentration tend to inhibit antibody binding non-specifically, so you need to be sure that your specific peptide inhibits while the non-specific one at the same concentration does not inhibit. 
We usually  perform competition with several peptide concentrations beginning at 50 ug/ml and down to 1ug/ml (or even lower if the antibody is of high affinity).

The antibody is pre-incubated with the peptide for a minimum of 2 hours and up to overnight, and than used for histology.

Good Luck,

Dorit Zharhary

From:  Carol Burden
Sent:  יום שישי 07 מאי 1999 23:00
Subject:  Peptide competition Assay

Hi ,
I'm trying to detect an antibody that has been prepared by an investigator and
is therefore largely uncharacterized.  We wish to verify the antibody by
performing a peptide competition.  Has anyone done this before with tissue??

Sincerely Thanks,

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