Antibody affinity in Western blotting

D.K. dk at
Wed Nov 28 07:59:20 EST 2001

"Martin Smith" <martin_smith at> wrote:
>I was wondering if antibody affinity for nonsequential epitopes on SDS
>mercaptoethanool linearized and electrotransfered polypeptides is a
>limitation of western blotting? Is it possible that unmarked polypeptides on
>final membrane cannot be recognized by certain serum antibodies for such

Yes, it can be a problem. You won't notice it when working with sera
because they will also contain plenty of "straight" epitopes, but it is
a common problem with monoclonals. Some MoAb work in IP but
do not work in Western. In this case attempts to renature antigen 
in gel before tranfer are sometimes successful. 


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