Membrane proteins extraction protocol - (Jul/08/2007 )
I am trying to look for the expression of EGFR in mammalian cells and was wondering if anyone could post a good protocol for extraction of mammalian cell membrane proteins. Any help is highly appreciated.
a protocol was mentionned in this forum previously. here it is :
Membrane fraction preparation
Make the following reagent:
0.1M K2HPO4 (pH 7.4)
(call this Homogenization buffer)
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.