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Immunoprecipitation - (Jul/24/2002 )

I am trying (without success) to pull down a transiently expressed c-myc epitope tagged protein.  From immunocytochemistry labelling, my protein is a soluble nuclear factor.  From the precipitation experiments I have done so far, I am getting several background (unwanted proteins) and not mine.  It is definitely present in the transfected cells (western blot analysis on total cell lysates).  Does anyone have any suggestions.  I have tried numerous washing buffers (RIPA, high salt etc) and am fast running out of ideas.  Can anyone  help?

Thank you



Sounds like your IP antibody is not very good.  Try a different antibody for comparison.  Santa Cruz Biotech ( has a ton of antibodies to choose from. Also Kierkegaard (spelling) Perry Labs (KPL) has a useful protocol manual with their IP kit.  Check out their website.


Some antibodies work very well in western-blot but are enable to recognize the native protein, so they are enable to immunoprecipitate correctly. Are you sure your antibody is usable  for immunoprecipitation ?


Does it need to be immunoprecipitation? You might consider doing a Far Western Blot or something like that if you are trying to find out protein-protein interactions. Other ideas include yeast two hybrid screen, phage display, GST pulldown, etc...but of course these require a lot more effort than simple immunoprecipitation, plus requires knowledge of molecular biology techniques.