Re: MS/digest
Ulf Hellman (ulf.hellman@licr.uu.se)
Tue, 05 Oct 1999 23:56:24 +0200
Dear Deb and others,
Since I was mentioned in your mail, I feel I could try to answer. The
protocol you describe sounds pretty standard to me - except for the use of
ethanol in destaining (or is it a mis-type?). I wouldn't believe there
could be any problem in performing an in-gel digest of those bands. This I
dare say because during the last 7 years we've been in situ digesting
Coomassie stained bands from almost everywhere ( well over 1000 samples)
and among those I am sure there is a large variation in the
staining/destaing protocols people have been using. Yet, when there was
fair amounts (a good blue band), we always get positive results at narrow
bore RPC, and last two years by MALDI (using some 0.5 % of total extract)
However, to really be certain, why don't you take the trouble of running a
test protein under exactly these particular (?) conditions and see what you
get?
Re your last comment, remaining Coomassie has really never bothered us; by
RPC it elutes at approximately 50 % acetonitrile, well after most peptides.
By MALDI, I can't say that we were bothered by the dye; on the other hand,
if the protein was seen by Coomassie, there is a lot of material, so the
dye doesn't harm?
What protocol are you using for in-gel CNBr? That of Susana Linskens?
Regards from Uppsala /Ulf
Ulf Hellman, Ph. D.
Prof.
Ludwig Institute for Cancer Research
Box 595
SE-751 24 Uppsala
Sweden
Phone +46 18 160423
Fax +46 18 160420
E-mail "ulf.hellman@licr.uu.se"
alt. "cykeldoktorn@hotmail.com"