In article <47382l$opc at netserv.unmc.edu>, dmlepley at netserv.unmc.edu (Denise Lepley) says:
>>Hello all! I was wondering what the consensus is for trying to use the
>peptide antigen to compete off non-specific primary antibody binding for
>western blot analysis. In my opinion, using the same peptide the
>antibody was raised against will compete off non-specific as well as
>specific antibody binding. So, in effect, adding peptide to the primary
>antibody solution is a meaningless control. What are your opinions on
I suppose that specific binding implies a high affinity binding while
non-specific binding is of low affinity. Therefore the high affinity
binding will decrease much more rapidly than the non-specific binding
when you add excess peptide while staining the western blot.
In my opinion adding peptide to the primary antibody solution is
a good control because when your band vanishes you can be sure that
that antigen has the same epitope as the peptide.
dirk.jochmans at chaos.kulnet.kuleuven.ac.be