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

Irit Rappley via methods%40net.bio.net (by irappley from scripps.edu)
Fri Apr 30 14:50:50 EST 2010


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
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