Detergent isolation of lipid rafts and caveolae-- explanation about components of 35% and 5% sucrose

Colleen Foley via methods%40net.bio.net (by colleenelizabeth.f from gmail.com)
Fri Apr 30 12:28:18 EST 2010


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


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