coimmunoprecipitation problems - (Jun/30/2005 )

Dear colleagues

I am trying to coimmunoprecipitate interesting proteins which interact with VSV-tagged Arrestin3 from 293 cell lysate using monoclonal Anti-VSV antibody. I can pull down most of Arrestin3 in Tris buffer with 1% Triton-X-100.

The problem is I can hardly pull down any other proteins instead of huge amount of cytoskeleton protein such as myosin and actin, which I think is nonsepecific binding to the IgG1.

Do you have any suggestion to get more coimmununoprecipitated proteins in the meantime get rid of cytoskeleton proteins.

Thank you very much

Terry

If it is nonspecific binding to the IgG preclearing with the IgG before you incubate with the primary antibody might help. I'm not an expert on co-IPs though.