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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