Audree Fowler
Department of Biological Chemistry
UCLA School of Medicine
Los Angeles, CA 90095-1737
Ph: 310 825-0222 or 5825-9842
FAX: 310 206-5272
Email: afowler@biochem.medsch.ucla.edu
Web:http://www.medsch.ucla.edu/som//biolchem/pmf
-----Original Message-----
From: abrf-request [SMTP:abrf-request@aecom.yu.edu]
Sent: Monday, March 30, 1998 9:46 AM
To: Recipients of ABRF List
Subject: CNBr cleavage
I was wondering if anyone can offer some advice on CNBr cleavages. I have
been trying to cleave my protein by adding a small CNBr crystal to my
protein in 70% TFA, but I'm not sure I am having much success. Either my
protein is not cleaved, or I think I am getting too many fragments when I
analyze my cleaved protein with MALDI-TOF. I usually just put my sample
in a
small tube and put this is a dark drawer for 16-18 hours.
I would also greatly appreciate it if anyone can help me with the
following
questions.
How long does a CNBr cleavage really take? I have read in the literature
it
is anywhere from 4-24 hours.
Do I need to remove the oxygen from my tube (covering with argon for
example), or is capping the tube sufficient?
Can adding too much CNBr lead to cleavages at other sites?
Is TFA the best choice to use considering I will be analyzing my protein
on
a MALDI?
Can 70% TFA lead to acid based cleavages by itself, and can formic acid
really lead to a reduction of disulfide bonds? ( I read a message about
the
formic acid on this discussion group a while ago)
Thanks a lot in advance
___________________________________________
NC State University
Department of Biochemistry
Phone (919)515-5786
Fax (919)515-2047