how to isolate membrane fraction from cells
Dr Engelbert Buxbaum
engelbert_buxbaum at hotmail.com
Thu May 20 12:50:03 EST 2004
> 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?
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.
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