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?
Sounds like your IP antibody is not very good. Try a different antibody for comparison. Santa Cruz Biotech (www.scbt.com) 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.