>
>
>Hi Rich,
>
>a ex post-doc of mine, Paola Dainese made exactly the same obervation as you
>with a
>variety of proteins including BSA. Firstly it turned out to be nothing to do
>with
>glycosylation as if often the case with odd orange coloured bands. At the
>time we
>had (a still do have) a fixation with the degree of staining and what the
>stain
>looks like in cross-section. Paola usually silver stained stained the bands
>for a long
>time and then destained until the desired degree of staining was obtained.
>Here one often
>sees the orange-brown colouration. We noticed that better yields were
>obtained with gels
>that were initially lightly stained as opposed to those taken to black and
>then destained.
>
>If you take a scalpel and cut a spot out and look at the cross-section of
>the gel you see
>that in the lightly stained gels only the two surfaces are stained, the
>middle part of the
>gel is colourless.
>
>In deeply coloured gels the staining went all the way through. Destaining
>low abundance spots
>that were originally orange gave the blue colour you mentioned though only
>on the surface
>where the staining was most intense.
>
>Dumb Guess: The colour is due to either Prussian Blue (presence of Fe in
>protein or solution) or Turnbulls (or Turnballs, my memory of my
>peripubescent years in the chemistry class is failing)
>blue which was due to Cu, Ag and various other metals. i.e. a lab dependent
>reproducible artifact.
>
>Recommendation: Use fluorescent stains, no need to fix (i.e. remove SDS and
>render proteins
>completely insoluble and refractive) and large dynamic range compared to the
>pathetic
>x50-100 linear range for silver. Try rubyPro or whatever it is called.
>S**pro orange and the
>other colours are us*less.
>
>Or- follow RD Smith's suggestion, do not stain or run 2D gels etc.- buy an
>FT-ICR-MS and do everything in the MS (detect, quantify, define mass and
>sequence).
I'll modify my answer a little and say, the methods shown by Ruedi
Aebersolds group are the way to go. We have a more generalised method which
can quantitate any gel spot (relative to an internal standard) without the
need for either isotopic labelling during growth, but only needing 2D gel
spots and post-gel labelling. As to rubypro, it works, the sypro did not. As
to why, hard to tell without the structures of the stains. This is a merely
rule of thumb observation. Sypro orange was very sensitive to the type of
protein being stained, even more so than silver.
PS A UV lightbox only costs 100$ US; a pair of mountaineers glasses about
the same again. Even if the stain does not suite you at least you can go on
holiday with an easy conscience.
ciao
Peter