Immunoprecipitation

Marieke R. Koedood Zhao rkoedood at bu.edu
Mon Aug 4 13:36:37 EST 1997


Joel Baguet (Joel.Baguet at ens-lyon.fr) wrote:
: Dear Netter's
: I try to co-immunoprecipitate partners of my protein from KCl lyzed 
: cellular extracts ... God it's not very easy

: The second problem: many non-specific bands appeared after ECL 
: staining, so how can I have the protein-partner binding still present 
: with these non-specific bands out of my immunoprecipitated sample. The 
: first antibody is the same antibody I used for immunoprecipitate the 
: protein and the partners. Can I biotinyle the purified antibody to 
: decrease non-specific bands?

don't have answers for all your problems, but here is a questions to 
consider - if your 1st Ab used ofr the Western is the same as the one used to
immunoprecipitate your proteins, then your 2nd Ab sill recognize this 1st
Ab on the Western. Let's try to rewrite it less confusing (I'm confusing
myself here :)). You use Ab-1 for immunopreciptating, after which the
proteins are run on a SDS-gel. the Ig-chains of Ab-1 are also on our
gel. Then you transfer and incubate the blot with Ab-1 again, so Ab-1
sticks to your specific protein. So far so good. However, when you add Ab-2,
it will stick to Ab-1, both the Ab-1 that is stuck to your specific protein
AND the Ab-1 Ig-chains that are also on the blot. so, to get rid of some
of that background you have to consider all the Ab-reactions that take
place.

Hope this helps a bit.

Marieke 



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