Emily:
When there are several disulfide bonds I usually use 3-5% mercapto-ethanol
(I usually use 1%). I also like to denature with 8M guanidine HCl, and heat
the protein at 80 degrees centigrade before digesting it.
Amina
Amina S. Woods, Ph.D.
NIDA Intramural Program, NIH
5500 Nathan Schock Drive
Baltimore, MD 21224
Tel: 410-550-1507
Fax: 410-550-2971
e-mail: awoods@intra.nida.nih.gov <mailto:awoods@intra.nida.nih.gov>
-----Original Message-----
From: Association of Biomolecular Resource Facilities
[mailto:abrf-request@aecom.yu.edu]On Behalf Of Simone Koenig
Sent: Monday, July 17, 2000 2:38 PM
To: Recipients of ABRF List
Subject: actin
Hello,
I am writing to see whether anyone has experience in digesting actin (42
kDa of monomeric bovine muscle). It contains 6 Cys residues, some of
which may be modified by oxidized glutathione (GSSG). My goal is to
identify the number and sites of glutathionylation. I have tried
in-solution digestion by trypsin, chymotrypsin, Glu-C, Lys-C and CNBr.
The sequence coverage is quite good. It seems that trypsin and Lys-C
gave the most fragments, however, there is a lot of incomplete digestion
giving rise to large peptides. I often see peptides in the 4000-8000 m/z
range that did not match any predicted masses. I thought that one
possibility is disulfide linkages, however, they did not match any of my
observed masses either. I would like to improve digestion until I get
more complete cleavage. I also looked into literature and there is not a
lot to refer to. I'd appreciate some help if anyone has experience in
actin.
Sincerely,
Emily Boja
email: bojae@nhlbi.nih.gov
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