RE>re: tryptic digest 8/23/2000
Ting-
I've seen these incomplete digestions, too, and have dealt with it by increasing the effective trypsin activity.
What are your digestion conditions? e.g. pH, temperature, enzyme/substrate ratio, enzyme source? You can increase your effective activity by changing the pH (8 rather than 7), temperature (37C rather than 25C), or increase the E/S ratio. In addition, I've found that Wako trypsin is better than Boeringer Mannheim trypsin in producing more complete cleavages at sites with multiple basic residues. (Does anyone know if Endo-LysC might do a better job at the KK site?)
Alternately, you can just identify the peaks in your peptide map, and accept getting variable ratios of these peaks. If you're doing a formal assay validation, document the effect of the above variables on the peak ratios, as part of the robustness studies.
Vernon
Vernon A. Shoup
Regeneron Pharmaceuticals
Rensselaer, NY 12144
(518)488-6012
vernon.shoup@regpha.com
--------------------------------------
Date: 8/23/2000 2:22 PM
To: VERNON SHOUP
From: Ting Wong
Hi, everybody:
On analysis of a tryptic map, I have the following fragments:
KT..........YKNPK
KT...........YK
T.............YKNPK
T..............YK
I know there is a lysine before the K in the sequence, so this is
apparently the result of incomplete digestion of the sequence:
KKT............YKNPK
Does anybody have suggestion on how to achieve complete digestion to
have a clean-cut map? Thanks in advance for suggestions.
Ting Wong
Biomira Inc.
This archive was generated by hypermail 2b29 : Fri Aug 25 2000 - 08:35:04 EDT