ProtSeq

James Farmar (jfarmar@nybc.org)
Wed, 15 Jul 1998 09:08:40 -0400

Dear ABRF Colleagues,
Does anyone know of a tried and true method/protocol using cyanogen bromide
to digest a low level amount (<1 pmol) of a protein in a gel slice or a
pvdf band? The method should be amenable to extraction of the peptides and
MALDI analysis after the reaction. Additionally, I would prefer a method
that avoided formic acid so as to avoid formylation of the cleavage products.
I want to determine the N and C termini of this known protein. The
resulting peptides will have a MW between 1000-6000 g/mole. I'm willing to
give the digest a try but the researchers have to go through a considerable
effort to get the protein.
My thoughts:
pvdf: CNBr/0.1M HCl/ACN or MeOH (for wetting?); extraction as per normal
digest
gel: will CNBr leave the polyacrylamide alone?

Thanks!

Dr. Jim Farmar
MicroChemistry
L.F. Kimball Research Institute
New York Blood Center
212 570-3128
212 570-3195