staining a chargeless protein
Dr Clinton A L Monfries
cmonfrie at ion.bpmf.ac.uk
Thu May 23 21:31:33 EST 1996
Phillip J Robinson wrote:
> Olin Anderson <oandersn at pw.usda.gov> wrote:
> >We are working with a 55 kDa polypeptide that has no charged amino acids.
> >Attempts to stain the polypeptide on PAGE gels has failed - tries
> >included commasie blue, amido black, silver, copper stain, Sypro,
> >anilionaphthalene sulfonate, and fluorescein isothiocyanate. The only
> >amino group is the N-terminal and it seems unreactive/blocked. The
> >only amino acids in the polypeptide are P, G, Q, Y, T, and S. Any
> >suggestions how to stain/detect this polypeptide in a gel?
> It seems simple enough to me. Here's two ideas for negative staining.
> However, you seem to have tried them already. I have to say that if
> both fail I also feel that you may have not got any "penetration" into
> the gel of your protein or one of the following really should work
> (shouldn't they?). Both the following do NOT depend on the charge of
> the protein to the best of my understanding, just on the fact that a
> protein occupies space in the gel.
> Try copper chloride (sulphate?) staining. It apparently stains the
> background, by turning it opaque, and leaves the protein
> clear/unstained. View on a black sheet of paper. I've tried it,
> seems more sensitive than coomassie, as originally claimed. I don't
> have the reference handy, but will email upon request.
> Alternatively, we have often observed that if one overstains a
> silver-stained gel, some proteins that were not stained show up
> beautifully clear against the overstained background.
> Anyone else out there with other ideas on such negative staining
> PS: I too am curious about the "natural" occurence of such a protein.
> Care to elaborate?
> Phil, Newcastle, Australia
Depending on how concentrated your protein is it may show up by staining
your gel in ice-cold 0.25M KCl. Proteins show up as a white precipitate
in the gel. I used to do this on preperative gels for the isolation of
b-Gal fusion proteins for immunization.
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