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Lipid raft extraction HELP! - (Jul/02/2010 )

i have been extracting lipid rafts for awhile now, but i seem to keep getting inconsistent result from my markers: caveolin and GM1.
Is there a standard protocol that would help with this. My lysis buffer is 10mMtris, 1%np40, 1mMedta, and 1:100 P.I inhibitors. I use 1% triton for the solubility.

-pizzapop-

what about the centrifugation speed? it must be very high to pellet those down. it is the last fraction I believe.


pizzapop on Jul 2 2010, 11:36 AM said:

i have been extracting lipid rafts for awhile now, but i seem to keep getting inconsistent result from my markers: caveolin and GM1.
Is there a standard protocol that would help with this. My lysis buffer is 10mMtris, 1%np40, 1mMedta, and 1:100 P.I inhibitors. I use 1% triton for the solubility.

-Curtis-

Pizzapop,
Did you ever fix this problem? I am also having problems with my Lipid raft extractions. I get consistent separation of lipid rafts but can't seem to get consistent recruitment of proteins to the lipid rafts. Any advice?

Curtis on Sat Jul 3 11:24:34 2010 said:


what about the centrifugation speed? it must be very high to pellet those down. it is the last fraction I believe.


i have been extracting lipid rafts for awhile now, but i seem to keep getting inconsistent result from my markers: caveolin and GM1.
Is there a standard protocol that would help with this. My lysis buffer is 10mMtris, 1%np40, 1mMedta, and 1:100 P.I inhibitors. I use 1% triton for the solubility.

-DrABK-