> I also have used BNPS-SK which gave 30%
>cleavage yield, but the elimination of BNPS excess is hard.
Dear Jose,
Sorry to be so tardy in responding, but it's just as well as I have
seen some very helpful responses to your query.
If you decide that you would prefer to use BNPS-skatole to cleave at
tryptophan residues, we found that removal of the excess reagent is quite
easy using ether. For example, if you are cleaving a solution-phase protein
in some percentage of acetic acid (I use 75%), when the reaction is complete
(which, we know, is not as complete as we would like), add a little water
(maybe 0.2-0.4 vol) and then some water-saturated ether (maybe 1-2 vol).
Mix and let settle (or centrifuge) and you'll see that the yellow color has
partitioned with the ether. Remove the ether, extract a couple more times
and then remove whatever ether remains by vacuum centrifugation. The
peptide and protein fragments remain in the aqueous phase.
The water is added before ether extraction because it appears the
ether extracts much of the acetic acid, too, and you need to bolster the
volume of the aqueous component. If your protein is blotted to PVDF and
some tryptophans have survived the electrophoresis/blotting process, the
PVDF can be added to the acid/BNPS-skatole solution. When cleavage is
"complete", remove the PVDF and do whatever you normally do to remove
peptides from the membrane, pooling the extraction solutions with the
cleavage solution, then ether extract. I'm sure you know that you may not
be able to elute large fragments from PVDF. I've used that to my advantage
on a specialized appilcation by cleaving a blotted protein with a Trp near
the N-terminus and then sequencing the PVDF after the N-terminal peptide had
been removed.
Good luck.
Carol
Carol Manley Beach, Ph.D.
Protein Sequence Analysis
Macromolecular Structure Analysis Facility
232 Combs Cancer Research Building
University of Kentucky
Lexington, KY 40536-0096
cmbeach@pop.uky.edu
(606)257-4932 (office)
(606)257-4475 (lab)
(606)323-3909 (FAX)