Coomassie staining of proteins on PVDF

Frank O. Fackelmayer Frank.Fackelmayer at uni-konstanz.de
Sat Nov 20 07:43:22 EST 1999



Thorsten Burmester wrote:

> I asked this question in bionet.molbio.proteins but it may be better
> here:
>
> After SDS-PAGE and Western transfer to PVDF I stained the proteins with
> 0.1% Coomassie Blue in H20 dd. Destaining with 50% methanol. To my
> surprise the band of interest was _not_ stained but remained white on a
> light blue background. Other proteins were stained well as expected. Any
> explanation?
>
>

Hi Thorsten,
We´ve had that staining bahavior before. I usually see the correct
staining, but every now and then it´s reversed.  There is no obvious
difference in either the procedure nor the staining solution (0.1%
Coomassie in 50% metOH) that yields the two different modes of staining,
though. I got the impression that if there is lots of the protein on the
membrane (e.g. one very intense band of recombinant protein) the staining
tends to be reversed. Lower amounts of the same protein stain "correctly".
Interestingly, when you stain for a longer time (or you keep the membrane
in the first batch of destainer for extended periods) , the reverse
staining sometimes reverts again, and you get "correct" staining.
I guess it might be the amount of bound SDS that makes the difference. As
SDS binds to the protein and makes it acidic, and acidic proteins stain
lousy with coomassie, it might be that the protein-bound SDS prevents the
coomassie from binding. After some time, the SDS has dissociated from the
protein, and the binding may then occur as expected. I should add that
that´s pure speculation, so if anyone has a more plausible explanation (or
even a reference...?), please let us hear about it.

Frank





More information about the Methods mailing list