Jun-
At 01:56 PM 8/28/00 -0400, you wrote:
>Would any experts out there share experience on pepsin digestion of a
protein?
>What acid should one use? TFA vs. Acetic acid vs. formic acid, etc. Does pH
>play a role in getting numbers of fragments? What incubation temperature
>should be used? Incubation duration? And how to quench the reaction?
>
*I've used 5% formic in the past. I doubt the type acid makes any
difference in specificity. Pepsin isn't very specific, as you probably
know, liking a variety of hydrophobic residues, but last time I used it I
recall many peptides starting with Phe. 37†C is what it likes in vivo. Do
you need to stop the digestion abruptly for any reason? If not, just
quench by lyophilization. Even if you do, flash freezing ought to be fast
enough to qualify for an abrupt stop, especially relative to a 24hr digestion.
Regards,
John
John Hempel, PhD Ph (412) 624 0161
University of Pittsburgh FAX (412) 624 4759
Department of Biological Sciences
Clapp Hall 301
Pittsburgh PA 15260 USA
email: hempel@psc.edu
http://www.pitt.edu/~biology/faculty/hempel.html
This archive was generated by hypermail 2b29 : Fri Sep 01 2000 - 16:10:38 EDT