lysis buffer for membrane bounded protein

[Dr Engelbert Buxbaum]

> I asked about the best lysis buffer for membrane bounded protein extraction.
> I would like to do SDS PAGE western blot and also native gel PAGE

That depends on what you mean by "membrane" (just the plasma membrane or
other membrane systems as well) and by "membrane bound" (only
transmembrane proteins or membrane associated proteis as well).

Assuming you mean the plasma membrane and any proteins that are bound to
it reasonably tightly you could lyse your cells in hypotonic buffer (30
min on ice in 10 mM Hepes/Tris of appropriate pH, with protease
inhibitors), remove cytosolic proteins by centrifugation (145000 g 30
min at 4 degrees), potter the pellet in a suitable buffer and then use a
simple step gradient to enrich plasma membranes (35% sucrose, 50000 g,
35 min in a swinging bucket rotor). PMs will be at the interface between
sample and sucrose. Collect, dilute 3 fold with buffer and spin the
membranes down (85000 g, 45 min). 

If you want to remove membrane associated proteins you can wash the
membranes with 100 mM Na2CO3 pH 11.5 30 min on ice, followed by
centrifugation. The pellet contains only transmembrane proteins, the
supernatant the membrane associated ones.

The pellet you can take up in any electrophoresis sample buffer you
like, for SDS-PAGE (or better CTAB-PAGE since you'll get crisper bands
from membrane glycoproteins) or for example blue native PAGE. Do a
protein determination first though, this is easier before detergents
have been added.