I had a poster at the last ABRF meeting in Durham about this. We found out that
while Sypro was probably one of the best to choose (from the point of view of
peptide recovery), it is not very convenient to work with it and, at least in my
hands, it is not sufficiently sensitive. I typically use the silver nitrate
stain with thiosulfate sensitization and WITHOUT glutaraldehyde. The trick to
get acceptable background with silver stain is as follows:
1) destain your gel (we use Farmer's reducer). There has been a publication
earlier this year on this.
2) reduce/alkylate in the gel. This seems to significantly reduce background
noise, probably from reactive species in the gel.
The known tricks, obviously, are still valid: digest in the smallest volumes
possible and, especially, use clean, NON-SILANIZED tubes (have you already seen
a LC-MS chromatogram full of silanized compounds? it really looks nice, except
there is no peptides in there ...).
Hope that it helps.
Axel
Roger Murphy wrote:
> Lisa,
>
> What about some of the fluorescent dye markers such as SYPRO Orange? I
> haven't used them myself but lots of my colleagues swear by them!
>
> Cheers,
>
> Roger
>
> At 12:44 PM 21-10-99 -0700, you wrote:
> >What is the best stain for sub-pmole amounts of protein to be in-gel
> >digested followed by Mass spec identification (MALDI followed by
> >LC-MS)? I have heard that Silver staining can be very bad and
> >produce no MALDI results. We haven't gone to sub-pmole yet so we
> >have just been using Coomassie and such.
> >
> >Thanks for your help.
> >
> >Lisa
> >Lisa Bibbs, Ph.D.
> >10550 N. Torrey Pines Rd.
> >SP3
> >La Jolla, CA 92039
> >
> >http://corefac.scripps.edu
>
> ------------------------------------------------------------
> Roger Murphy, Ph.D.
> Biological Production Facility
> Ludwig Institute for Cancer Research
> Austin & Repatriation Medical Centre
> Studley Road,
> Heidelberg, Vic. 3084
> Australia.
>
> Tel 61-3-94965463
> Fax 61-3-94965436
> Email Roger.Murphy@Ludwig.edu.au
-- --------------------------------------------------------------------------------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 Canada
tel. + (514) 428-3428 fax + (514) 428-4900