[Protein-analysis] Re: Immunoprecipitation

Dr Engelbert Buxbaum engelbert_buxbaum at hotmail.com
Fri May 26 06:22:29 EST 2006


atahualpa wrote:


> 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. 


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