Need prototocol for membrane fractionation from mammalian cells - (Aug/24/2006 )

Hi,

I want to check the locolization of a protein by using different fraction of cells, now I 'v got the method of nuclear fractionation, but still miss the membrane one... Could any one kindly recommend a protocol or some articles related to this method? Thanks in advance!

Hi
I used to do fractionation of the cytosol and membranal parts of mouse hippocampus.
Here's the protocol I used:
I homogenized the cells in a sucrose buffer: 250mM sucrose, 2mM EDTA, 2mM EGTA, 10mM DTT plus protease and phosphatase inhibitors.
After homogenizing - 30min on ice
Centrifugation 1hr 4C 45,000rpm
Supernatant is cytosolic fraction
Suspend pellet in same buffer with 1% Tritonx100. 1hr on ice and repeat centrifugation. Supernatant includes the membranes.
Hope this helps