Immunoprecipitation

Joel Baguet Joel.Baguet at ens-lyon.fr
Mon Jul 21 09:29:35 EST 1997


Dear Netter's
I try to co-immunoprecipitate partners of my protein from KCl lyzed 
cellular extracts ... God it's not very easy

The first problem: protein-A sepharose (Pharmacia) alone without any 
kind of cellular extract or antibody or serum give me a very beautifuk 
band (42 kDa protein A I suppose) and many low molecular weigth bands 
after boiling 95°C 3 min and immunostaining of the membrane by ECL 
(first and second antibodies)

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?

The third problem is: what are the best buffer and what are the 
centrifugation speeds for the washs of the proteinA sepharose washs?

Thank You



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