Silver staining problem
jbs at sgibio.chem.temple.edu
Wed Feb 14 14:09:34 EST 1996
Eric Sarnighausen wrote:
> I am analyzing patterns of cell wall proteins by two dimensional
> electrophoresis. Unfortunately, the most prominent protein of 23 kDa
> (that I am extremely interested in) is not silver stained at all (using
> the acidic method).
> As I know this protein to be glycosylated, I used the combined Alcian
> blue/silver staining method as described by Jay et al. 1990 (Anal.
> Biochem. 185: 324-330 - I did not use the PAS-method as I want to stain
> both glycosylated and not glycosylated proteins). However, this did not
> alter the (one dimensional) pattern of detectable cell wall proteins,
> the 23 kDa protein still not being stained.
> This protein also shows a strange behavior when I use Coomassie-
> staining, as (only) sometimes it does not bind the dye very efficient-
> ly. I have not yet figured out the reason for that.
> Has anybody found the solution to a similar problem???
> Any help/suggestions are greatly appreciated.
> Kind rgds
> EricWe found several years ago that many of the protein stains were non-
linear - apparently because they react with different portions of the
molecules. Coomassie Blue, for instance binds by charge, which is why it
is used at low pH, but it also is eluted by organic solvents, so it is
also binding by hydrophobic interactions. The only stain that we found
that was proportional to the length of the backbone was good old Amido
Black, used at low pH. For certain proteins, Amido Black gives a
stronger signal than Coomassie. You may have too little to detect. We
can see a spot of 1 microgram of protein. See Anal. Biochem 166:49-54.
Joel B. Sheffield
Biology Department, Temple University
Philadelphia, PA 19122
jbs at sgibio.chem.temple.edu or
(215) 204 8854
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