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I have a following problem with immunoprecipitation:
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). Could it be that lysis
buffer (contains 1% NP40) affects binding conditions of those two not
affecting binding of the "original" protein?
I would appreciate all suggestions.
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.
On Fri, 16 Feb 2007, . wrote:
If I understand you correctly, could it be that your antibody will detect
the reduced forms in western blot, but not the native forms?
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