Quick and stupid question: what types of proteins end up in both the soluble and insoluble fractions when cells are scraped in buffer with Triton X-100? I thought membranes (with their accompanying proteins) ended up in the pellet, and everything else was in the supernatant, but I can't find anything to confirm this.
Also, if anyone has good references for this type of stuff, please let me know. : )
1 reply to this topic
Posted 22 October 2003 - 04:38 AM
Good references are any by a guy called Kai Simons (for rafts). In my experience, if you have your protein in both fractions, there is a problem with the protocol. This happened to me for 2 years until we realised that though we were doing everything on ice - the scraping, centrifuging, etc, it was not good enough. EVERYTHING has to be done in a cold room, including keeping the solutions in there so they're ice cold. Using a cell type that expresses caveolin-1 really helps as that should be a control for complete insolubility. Na/K ATPase alpha-1 subunit should also be completely soluble, although cells invariably express either one or the other. Other than that, the amount of cells or protein in the prep also affects solubility as the Triton replaces the fat in the membrane. Look up any of the MAL family papers for extraction protocols. If that doesn't help, I'll send my protocol.