Hi Colleen,
By definition, lipid rafts are insoluble in Triton X-100. That would
be the reason for including the detergent in your solutions. I am not
familiar with detergent-free protocols, so I can't help you with that.
Irit
On Apr 30, 2010, at 10:28 AM, Colleen Foley wrote:
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
[email protected]
http://www.bio.net/biomail/listinfo/methods
_______________________________________________
Methods mailing list
[email protected]
http://www.bio.net/biomail/listinfo/methods
