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?
I think that your the c-myc epitope does not locate at the surface of your fusion protein. That may be the reason you could detect it by western blot analysis but not by immunoprecipition.
Try isolating the nuclear fraction (try a kit) and then use PBS rather than RIPA since it is a milder buffer for washing you beads. RIPA many degrade your protein hence the presence of background from your western.