sarita malik wrote:
>> i have a very simple Query
> how do u estimate protein concentration of a whole cell protein sample
> denatured in lammeli buffer for performing PAGE. i tried Lowrys method
> and Brad ford but both methods gave inconsistent results. when compared
> compared with electrophoresis of a known protein sample.
Laemli-buffer contains SDS (a detergent) and bME (a reducing agent)
which interfere with many protein assays. If your sample does not
contain dye you can try to use UV-absorption. Otherwise you can either
bind the protein to nitrocellulose membranes (spot blot), stain and then
elute the bound dye with NaOH for photometric determination or you can
precipitate the protein either with TCA/DOC (Bensadoun & Weinstein) or
Chloroform/Methanol (Wessel & Flügge). The precipitate is then
reconstituted in a medium compatible with your favourite protein assay.