how to isolate membrane fraction from cells

Hi there,

For my project I need to make clean Westren blots. The only way I have not
tried yet is to purify the membrane fraction from my cells (BAF/B03, which
is a murine precursor B cell line). The protein I am looking for is the
human growth hormone receptor, which is a membrane bound protein.
Can somebody please tell me how to isolate this membrane fraction? I have
heard about specialised kits (but without references) and some techniques
using high speed centrifugation.
Please help me out.

Thanks a lot,
Brenda

brenda wrote:


If you have a good antibody, your westerns should be fine directly from
whole cells solubilised in SDS sample buffer. Note however that
non-glycosylated precursor proteins may be present.

Also present may be degradation products, especially if you left out
protease inhibitor coctail (in my hands, 5 mM 6-aminocaproic acid, 100
uM PMSF (added immediately before use from 1 M stock in methanol or
i-propanol, *not* DMSO), 100 uM EDTA, 100 uM benzamidine, 1 uM
leupeptin, 1 uM pepstatin work quite well).

Membranes are usually isolated by fractionaed centrifugation, a process
described in detail in several papers in Meth. Enzymol. 31 (1974).

A quicker protocol involving osmotic lysis is described in Orlowski et
al., Cancer Biochem. Biophys. 16 (1998) 85-111.