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IP of endogenous protein - (Apr/04/2005 )

I am able to detect an endogenous protein (CKIIbeta) by western blot (50ug of total lysate), but unable to immunoprecipitate it with the same antibodies. I used HeLa and 293 cells and the cells were lysed in a buffer containing 0,1% NP-40. I tryed two differents antibodies with protein G agarose or protein A. In all the cases it didn't work. But I can detect my protein in the supernatant of the IPs (before the washes) at the same level as the total lysate, which make me think the antibodies don't recognise well the protein in the lysate. As it is a subunit part of a complex do you have any suggestion (lysis buffers etc.) that could help me?




It sounds like your antibodies have been raised against peptides and therefore may not recognise 3D structures. A couple of options, find an antibody raised against a domain of the protein of interest. You could also try adding a little SDS (0.1%-0.5%) or reducing agent to your lysis buffer and therefore hopefully break down some of the secondary, of course the problem is if you are looking for interactions or function then you will most likely lose them.

Sorry can't be more help