western blots

Unknown Unknown
Fri Nov 3 07:18:51 EST 1995


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 
>this?  

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

dirk.jochmans at chaos.kulnet.kuleuven.ac.be







More information about the Methods mailing list