Bradford Protein Assay in membrane crystals

Gerd Hoffmann gerd.hoffmann at nambition.com
Fri Jan 28 06:55:27 EST 2005


Dr Engelbert Buxbaum wrote:
> Gerd Hoffmann wrote:
> 
> 
>>Hi,
>>
>>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. 


Thank you for your answer! In the moment we want to try the BCA method.
As a "fallback" we try the other experiments.
Zhank You.

Gerd Hoffmann



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