Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum

Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum (http://www.molecularstation.com/forum/)
-   Protein Forum (http://www.molecularstation.com/forum/protein-forum/)
-   -   Bradford Protein Assay in membrane crystals (http://www.molecularstation.com/forum/protein-forum/26542-bradford-protein-assay-membrane-crystals.html)

Dr Engelbert Buxbaum 01-26-2005 03:45 PM

Bradford Protein Assay in membrane crystals
 
Gerd Hoffmann wrote:


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.

Gerd Hoffmann 01-28-2005 11:55 AM

Bradford Protein Assay in membrane crystals
 
Dr Engelbert Buxbaum wrote:


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


All times are GMT. The time now is 06:11 PM.

Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2014, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved

Page generated in 0.08990 seconds with 11 queries