John,
We use essentially a modified procedure of Ulf Hellman et al. changing the
coomassie extraction by washing with 60 ul 10 mM AMBI(pH 8.2)/50% ACN/10 mM
DTT (trypsin), or 50 mM Tris(pH 9)/50% ACN/10 mM DTT (endolys-C) and
incubate at 50C for 30-45 min. Remove the liquid, and add 60 ul the
buffer/ACN containing 50 mM IAM. Incubate 30 min. in the dark at room temp.
Remove liquid and add 60 ul of the DTT solution, vortex, remove liquid, and
speedvac to dryness. The few times we've done this, we got near
quantitative conversion of cysteines. If you do MS down at the level of a
couple hundred femtomoles, you will probably run into keratin. Like some
others, I believe it's coming from the DTT and so we are working to replace
the DTT with TCEP to avoid this contaminant and IAM with N-iPr-acrylamide
for the Edman HPLC, but so far we haven't enough data to say it works.
Let me know how this works out for you.
regards,
Gary
P.S. Enjoyed the cardiac hypertrophy paper.
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Gary M. Hathaway, Director
PPMAL - Protein/Peptide Micro Analytical Laboratory
California Institute of Technology
139-74, Division of Biology
Pasadena, CA 91125
http://www.cco.caltech.edu/~ppmal
email Gary: hathaway@caltech.edu
email facility: ppmal@caltech.edu
phone: lab (818) 395-6388 or office (818) 395-2769
FAX (818) 449-3414
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