Co-IP of secreted proteins - (Jan/03/2011 )
I try to perform co-immunoprecipitation with cell culture supernatant as my two proteins of interest are secreted. I've read suggestions for IPs with cell media. One is to concentrate the media via centrifugation filters. Is that also applicable for co-IPs? Does the centrifugation interfer with the interaction of my proteins? Are there other more sensitive concentration methods? Another problem is that one of the proteins is rather unstable and is a protease. Well, actually that's two more problems... That's why I don't know if I can use protease inhibitors...
Any suggestions? I'm thankful for any advice.
if the interaction involves the protease active site then you are correct in assuming that you can't use protease inhibitors.
if the binding is at a different site then you may be able to use an inhibitor that blocks the active site without significantly altering the conformation of the protein.