2 replies to this topic

[nsampson]

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

Natalie

nsampson@hgmp.mrc.ac.uk

[IjHuang]

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.  

[labslave]

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.