I am performing cytoplasmic/nuclear fractionation of HeLa cells with the objective of performing IP using antibody-conjugated magnetic beads on both fractions. I need to make sure I am not disrupting protein-protein and protein-RNA interactions too much, but I also need to make sure I am actually getting the contents of the nuclei.
I heard that NP-40 permeabilizes the nuclear membrane without breaking it; currently at 0.5% NP-40 I am having a hard time seeing my target protein in the control Western blot although I know for sure that it is in the nucleus and that it is not a blotting problem. How much NP-40 can I put in without interfering with downstream IP? And do you have any other suggestions?
My favorite co-IP buffer contains 50mM Tris-HCl (pH 7.5), 150mM KCl, 0.5% NP-40, 0.5 mM EDTA (pH 8.0), 1mM DTT, 0.1% SDS, 2.5mM sodium pyrophosphate, 1mM beta-glycerophosphate, 1mM Na3VO4, and protease inhibitors. If you subject your lysate to excessive sonication, you will release all of the nuclear proteins. I suspect even in sonicated samples there will be fragments of RNA to allow protein:RNA interactions.
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