Number Ten Ox
scottk at nuclease.berkeley.edu
Thu Oct 15 21:33:09 EST 1992
In article <92Oct06.063852.21808 at acs.ucalgary.ca>,
kfvanomm at acs.ucalgary.ca (Fintan VanOmmen Kloeke) writes:
|> I have a question that follows from the Coomassie one.
|> At the moment I am running protein gels on Pharmacia's PhastGel
|> system. It seems that high sensitivity silver stainning works
|> better if the gel is Coomassie dyed first.
|> Does anyone know why this is so? The Pharmacia reps didn't have a
|> Thanks in advance.
Well, I can't say I know *why* this is true, but it is a fact that
double-staining (coomassie followed by silver) is better for many
applications than silver staining alone. A paper in BioTechniques
a couple of years back (1990, vol 9 no 5, p 532) claimed a 40-fold
increase in silver stain sensitivity when gels were first stained
with a colloidal coomassie stain. In my hands, 2 to 5 fold is more
like it. Your mileage may vary. The double staining also
evens out staining of some proteins, i.e. some proteins which are
stained poorly or not at all with silver are stained well by
the double stain protocol.
This brings up another little tidbit: using colloidal coomassie to
stain gels. The best (only?) reference for the technique is Neuhoff
et al (1988) Electrophoresis 9 pp 255-262. They report *coomassie*
stain sensitivities down to 1 ng and lower. Yes, nanogram. I've
easily detected 10 ng in 0.75 mm thick SDS minigels.
Basically, the protocol involves staining with coomassie in the
presence of ammonium sulfate and methanol. In an ammonium
sulfate solution, Coomassie forms a colloid which is excluded from
the gel, while the methanol releases some of the dye into a molecularly
dispersed form which can diffuse into the gel and bind the protein.
The result is staining with essentially no background; no background
means no destaining, which means no loss of stain from the
protein. Check it out.
Integrated Separations Systems sells a kit for those
who don't want to play with optimizing the system for themselves.
____ Scott Keeney, DNA repair-queer ____
\ / scottk at mendel.berkeley.edu \ /
\/ Biochemistry and Molecular Biology \/
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