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

Dr Engelbert Buxbaum via methods%40net.bio.net (by engelbert_buxbaum from hotmail.com)
Fri Apr 30 15:14:44 EST 2010


In article <mailman.591.1272653145.25217.methods from net.bio.net>, 
colleenelizabeth.f from gmail.com says...
> 
> 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?

The idea is that "detergent resistant membranes" (usually equated with 
rafts) survive the initial solubilisation of the membranes in Triton. I 
can not see any reason to have detergent in the gradients afterwards, 
when these DRM are purified. On the contrary, I would leave it out, so 
that the detergent present during solubilisation is stripped from the 
membranes as they pass through the gradient. Remember: Just because 
Triton does not solubilise the membranes doesn't necessarily mean that 
it can not partition into the lipid phase or bind to the proteins 
present. Either would likely change the properties. 

The situation is completely different with solubilised proteins: As they 
are not water-soluble by themselfs, you need to have detergent present 
at the cmc during all purification steps. Otherwise, the proteins would 
aggregate.

These two situations should be kept mentally well separated. So, 
incubate your membranes with Triton, then load the mixture onto 
gradients w/o Triton to purify any membranes that survived the 
incubation. 

BTW, when you keep samples on ice, why work in the cold room? Cold rooms 
chill the operator, not the sample. In particular (and contrary to 
popular believes), cold rooms can not keep (clinical) centrifuge rotors 
cold during operation. If you want to do a "crap-spin" with your sample, 
do it in a refrigerated centrifuge.


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