Just wanted to add my $0.02 to this, and to relate our experience.
Before we got into the in-gel digestion game, we always used a commercial silver stain kit from Daiichi, which gave excellent sensitivity and image quality. Unfortunately, in-gel digest from bands with this stain gave lousy results, with recoveries much less than 10%. These bands were typically stained all the way through the gel.
The Mann 1996 protocol gave much better recoveries, and bands were always stained only on their surfaces. The most significant difference between the two stains is that the Daiichi uses a basic silver-ammonia step, while the Mann uses just silver nitrate in water. For a discussion and comparison of the mechanisms of silver staining, I recommend articles by T. Raibilloud (Electrophoresis 13 429-39; and Electrophoresis 11 785-794).
We also recommend the Mann stain (which is a slight modification of an earlier protocol by Raibilloud) to collaborators. It is our belief that the greater silver staining observed in reagents like the Daiichi kit works against recovery of peptides from in-gel digests. The only critical step that we have seen in the Mann protocol is the water wash after the silver nitrate step. Over-washing at this point can remove too much of the silver and result in poor staining.
Yours,
Elliott Nickbarg
Genetics Inst. Inc.
Cambridge MA 02140
>>> Deb McMillen <mcmillen@morel.uoregon.edu> 06/03 6:51 PM >>>
Hi, all,
I have a researcher here who has silver stained a gel (using the Mattias
Mann 1996 protocol)--his band looks a bit odd (this gel is 1.5 mm thick)
in that the staining looks like it is on the surface of both sides of the
band but not through the thickness of the gel--has anyone seen this
before?
Thanks,
Deb McMillen
Institute of Molecular Biology
University of Oregon
EUgene OR 97403
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