Here are my (at the moment) two favorite stains for 2D gels:
Colloidal Coomassie, with an average sensitivity of maybe 20-50 ng per protein;
it is really easy to prepare (it is a 2-steps procedure. I am using a kit from
Novex) but it is rather slow (the destainig takes forever). Other problem: the
stain is light blue and is difficult to scan. I typically use that stain for
visualization of proteins for in-gel digest (the colloidal Coomassie is
extracted in organic solvents). I am using a capillary LC-MS/MS system for
identification and I know that I can handle pretty much everything that is
stained by this dye.
Silver Nitrate according to Rabilloud, with an average sensitivity to the low
ng/high pg per protein. This is a bit more work but gives really nice pattern.
It is even somehow linear if you care not to develop too long. This sensitivity
is usually all what you really need for most of your work. However, because you
are treating your proteins with formaldahyde and glutaraldehyde, peptide yield
for in-gel digest is quite bad.
I tried Sypro orange - with very disappointing results. First it is really
expensive, second -at least in my hand- does not give much better results than
colloidal coomassie. I tried a improved protocol as described in the Siena
meeting (fix 2h in 40% EtOH, 2% acetic acid, 0.01% SDS for 2 h, wash for 2h in
2% acetic acid, 0.01% SDS, stain for 3-4 h with 2% acetic acid, 0.01% SDS,
1:5000 Sypro Orange, optional destain in 2% acetic acid, 0.01% SDS ; procedure
from JP Malone, Monsanto Company) but I was not very convinced by the results
neither. Plus there is still the problem of cutting a spot that you do not see
under normal light ...
You may want also to have a look at a very nice review of stains in "The
Scientist" vol 12 #10 (May 1998) pp 16-23 (the article is entitled: Dye hard)
by Michael Brush.
Good luck
Axel
grant.ra@pg.com wrote:
> We run a lot of 2D gels (mini and large format). Often Coomassie blue
> stain (even the most sensitive methods) does not give us enough sensitivity
> to visualize as many proteins as we want to see, so we need to silver stain
> our gels. This is a rather time-consuming and often not-very-reproducible
> process, especially if you have 10-20 gels to stain.
>
> What other staining methods are people routinely using that are in the same
> range of sensitivity as silver? How reproducible are they? If you use
> fluorescent stains, how do you see the spot while you are cutting it out?
> Assuming that there is enough mass in a particular spot, can the proteins
> be identified using peptide mass mapping or other MS methods? i. e., is
> the stain compatible with identification by these methods?
>
> Thanks!
>
> Ray Grant
> The Procter & Gamble Co.
> Cincinnati, OH
>
> grant.ra@pg.com
> 513-627-2179
-- Axel Ducret, Ph.D. Senior Research Biologist Merck-Frosst Canada Inc. Dept. Biochemistry and Molecular Biology P.O. Box 1005 Pointe-Claire-Dorval PQ H9R 4P8 Canadatel. + (514) 428-3428 fax + (514) 428-4900