I'm using a non-denaturing lysis buffer (50mM TrisHCL pH7.5, 150mM NaCl, 1%NP40/IGEPAL +ROCHE protease inhib., 1mM PMSF(fresh), 10mM B-glycerophosphate(fresh)) to lyse cells (30min on ice, then clarified). I plan on incubating the lysate with an antibody overnight @4*C(rotating) followed by protein-A conjugated agarose beads, then western. (No preclearing)
Im currently trying to optimize the assay and would like to know if anyone has an antibody (like B-Actin or vinculin) that they know works for IP, so I can optimize the assay before using lots of virus infected cell samples.
Im using Vero cells (monkey), but if you've used the antibody on any human cell line I also have 293T and several hu cancer cell lines.
Do you have any monoclonal antibody in your lab? If so, I think it may be worth trying to use that mAb to pull down its own natural ligand? Also, have you looked into the literature to see which some people use as their negative/positive control in IP? Also, I believe that particular condition is usually needed for a given protein, so even if it works in the protein you used in optimizing the assay, that optimized condition may not work for your experimental sample.
Yea I know there are antibodies in the literature that say they are good for IP, but I was looking for an antibody that a fellow grad student or post doc can personally vouch for as a control ie GAPDH, Vinculin, Actin, that have given consistently good pull downs.
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