Bradford Protein Assay in membrane crystals
Dr Engelbert Buxbaum
engelbert_buxbaum at hotmail.com
Wed Jan 26 10:45:18 EST 2005
Gerd Hoffmann wrote:
> we want to try to determine protein concentration in a solution
> containing fragments of "membrane-protein-crystalls". We
> want to try this with a bradford assay.
Any lipophilic or amphiphilic compound will interfere with Bradford,
including lipids and detergents.
To remove the large fragments you may have to do protein precipitation
either with chloroform/methanol (Wessel & Fluegge) or with TCA using
desoxycholate as carrier (Bensadoun & Weinstein). There are a number of
alternative methods (e.g. phenol/ether, calcium phosphate or certain
dyes) which have not found widespread application. The purified protein
is then dissolved in alkaline solution and determined either by the
Lowry et al. procedure or with BCA (which is probably what the previous
poster was referring to as "Pierce assay").
Alternatively, you can dot blot your sample onto a PVDF membrane, wash
thoroughly and then stain the bound protein with Coomassie. The amount
of stain bound to a spot can then be determined either with a flat bed
scanner and quantitation with NIH-image, or by extraction of the dye
with alkaly and photometric determination. In particular the scanner
route is very quick and reasonably precise.
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