I cannot draw conclusions on the basis of one experiment,but we used about
6N CNBr in acetonitrile,and it kept for
quite a while at r.t.Having such concentrated solution,it is possible to
add a small volume.The acetonitrile does not
interfere.
Henriette Remmer <remmer@ux1.cso.uiuc.edu>@aecom.yu.edu> on 10/16/2000
10:16:18 AM
Sent by: Association of Biomolecular Resource Facilities
<abrf-request@aecom.yu.edu>
To: Recipients of ABRF List <abrf@aecom.yu.edu>
cc:
Subject: Re: Protseq:cyanogen bromide cleavage
Dear Amanda:
The following method works well in solution, in suspension and on pvdf, it
should also works in-gel, although extractions of the fragments may be a
problem.
Reduction: dissolve protein in 0.2 Ammonium bicarbonate solution, add 1-5%
(v/v) beta-mercaptoethanol. Blow N2 over the tube to displace oxygen, seal
the tube , incubate at room temp. 18 h.
Cleavage:
Dry sample completely under vacuum, warming if necessary.
Redissolve sample in formic acid , add water (or other solvent) to make the
acid 70%(v/v) finally; add CNBr (equal weight to protein). Seal the tube
and incubate in the dark 24h.
Terminate the reaction by drying down under vacuum. Store sample at -20C or
use immediately.
You may weigh out a certain amount of CNBr and make a concentrated solution
in water (under the hood) in order to add the CNBr in a minimum volume.
Use only white CNBr crystals, dispose of excess solution by reacting it
with bleach (caution! stay under the hood).
Good luck!
Henriette
Hi everyone,
I need a method for generating peptides by cyanogen bromide from gel
pieces. Does anyone have a good method and also what safety precautions
should I take - weighing out, drying down, disposal etc.
Thanks
Amanda Hall-Griffin
Professional Officer
Newcastle Protein
University of Newcastle
Ph (02) 4921-7299
Fax (02) 4921-6903
Henriette A. Remmer, Ph.D
Director of Protein Services
Protein Sciences Facility at the Biotechnology Center
University of Illinois
311/315 Noyes Laboratory, Box 62-1
505 S. Mathews Ave.
Urbana,IL 61801
Tel: (217) 333-4695 (Lab) (217) 333-3841 (office)
Fax: (217) 244-1142
homepage: http://www.life.uiuc.edu/biotech/protein_sc.html
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