Norman-
It is not clear what you plan to do after visualizing your band, and how you
stain your gel can have serious ramifications for some downstream analyses.
In general, for the abundance range you're considering, I find that silver
staining (several protocols are available) is routine and reliable and
sensitive to about 1 ng. A colloidal coomassie stain (Novex/Invitrogen
sells a kit) is almost as sensitive and requires much less hands-on labor
(although you have to wait longer for the gel to de-stain fully). Many
prefer the SYPRO stains (i.e. Orange, Red, Ruby) which are reported to have
the same sensitivity as silver. Reverse-staining with ZnCl2 and imidazole
(Fernandex-Patron et al., Biotechniques 12:564) is rapid but does not last
long. I do not rely on these last two as they have given me mixed results
(what have other people found?). If you're expecting 50 ng in your band, I
suggest either the silver or colloidal coomassie stain (or even standard
old-fashioned coomassie, which is not as sensitive as any of the others I
have mentioned). However, the sensitive silver stain is likely to give you
a very high background if you have other abundant proteins in your sample.
If you plan to analyze the protein(s) by mass spectrometry (i.e., after
in-gel digestion), then you need to be careful not to fix the protein(s) too
much. Colloidal coomassie works well for this, or you can use a
low-fixation silver stain protocol (Shevchenko et al., Anal. Chem. 68:850)
coupled with a silver removal step (Gharahdaghi et al., Electrophoresis
20:601). I have found the latter to be the most reliable and robust for
subsequent in-gel digestion and peptide analysis by mass spectrometry.
Cheers,
David
__________________________________________________
David B. Friedman, Ph.D. (303) 315-4712
Dept. of Cellular and Structural Biology, "I am not a number,
University of Colorado School of Medicine I am a Friedman!"
UCHSC, B-111, SOM 4538
4200 E. Ninth Ave.
Denver, Colorado 80262
-----Original Message-----
From: Norman Watts [mailto:WATTS@calvin.niams.nih.gov]
Sent: Wednesday, December 13, 2000 6:15 AM
To: Recipients of ABRF List
Subject: Misc, SPS-PAGE, sensitive protein stain needed
Hi all,
What is currently considered to be the most sensitive and
dependable stain for detecting protein bands on SDS-PAGE
gels? I expect ca. 0.05 ug/band.
norman
-------------------------------------------------------
Norman Watts, PhD.
Lab. of Structural Biology
Building 6, Room B2-34
6 Center Drive MSC2717
NIAMS, National Institutes of Health
Bethesda, MD 20892-2717
U.S.A.
E-mail:watts@calvin.niams.nih.gov
Phone: 301-402-3418
Fax: 301-480-7629
-------------------------------------------------------
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