question on membrane extraction protocol - (Oct/16/2007 )

Hi

I read the following protocol for membrane fraction separation in a previous post. I was wondering if omitting the final spin of 100000 (or 50000) rpm would yield a membrane AND cytosolic extract or are nuclear proteins present as well (probably so, considering the sonication step?)...
thanks


Make the following reagent:
0.25M sucrose
0.1M K2HPO4 (pH 7.4)
1mM EDTA
(call this Homogenization buffer)
keep chilled

Remove cells from culture and aspirate media. Wash cells twice with PBS (cold)
Scrape cells in PBS and spin 10min 2700 rpm Aspirate PBS and resuspend cells in 500ul (for a 100mm plate) of the homogenization buffer and add protease inhibitors. Sonicate cells in the homo. buffer. The cells should go from white, cloudy suspension to a opalescent solution.

Spin 15 min 2000 rpm. Remove supernate and discard pellet. Spin supernate 15 min 100,000 rpm or alternatively spin 1hr 55,000 rpm. This time keep the pellet as this is your membrane prep. Discard the supernate and resuspend the pellet in an appropriate buffer.