I will be using a lysis buffer consisting of MBS, 1% Triton X-100 (with protease inhibitor) to isolate cavelae and lipid rafts from BHK cells.
I have found several papers using this method that use 35 and 5% sucrose in MBS without Triton X-100 and another (from the book Methods in Membrane Lipids, can be found on google books) in which the 35 and 5% sucrose solutions are in MBS +Triton X-100. I will be comparing the results of the Triton isolation to a detergent free isolation with Sodium Carbonate lysis buffer. In the detergent free isolation the 35 and 5% sucrose solutions are in MBS + sodium carbonate. I am going to put the 35 and 5% sucrose solutions for the detergent (Triton X-100) isolation in MBS + Triton X-100. But I was wondering if anyone had any thoughts on why there are protocols out there that either leave out the fact that Triton X-100 is in these sucrose solutions or leave it out of the solutions for a reason. I'm assuming it has something to do with the fact that lipid rafts are insoluble in detergents such as Triton X-100 but I want to understand the "how and why" of this from a biochemical standpoint better. Also, is it standard procedure to perform the isolation in a 4 deg C cold room, with everything on ice? -Thank you. Colleen _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods
