2 replies to this topic

[pizzapop]

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]

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.

[DrABK]

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 03 July 2010 - 03:24 AM, said:

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.