> When I develope western of the cell extract with the antibodies for the
> protein I am dealing with I see the protein that I expect and two, much
> thicker, bands higher. I want to immunoprecipitate them and go for MALDI. I
> have run immunoprecipitation of the cell extract with the same antibodies
> and I run gel for coomasie staining. I can see on it very nice band of my
> protein and nothing more beside background. It seems that IP works since I
> can catch my protein, but where are the other two guys? Why they are not
> caught in IP although very nicely detected on western with same antibodies?
> The only reason that comes to my mind is that western is developed in
> conditions different from IP (milk vs lysis buffer).
There is a much more important difference: In a western you have
proteins denatured by SDS and sulphhydryl-reagent. In IP you work with
more or less (depending on IP buffer composition) native proteins.
Antibodies directed against conformational epitopes rather than a
sequence will react very differently under these conditions. This
problem is larger with monoclonal antibodies than with antiserum.