Many cyanogen bromide cleavage protocols fail to cleave ubiquitin because of its stability in acid. The following protocol has proved effective for cleaving the ubiquitin mutant I36W P37M F45W.
- Dissolve ubiquitin in 70% formic acid to a concentration of 1mg/mL.
- Add CNBr to 20mg/mL. For 3M CNBr in dichloromethane (under the hood), this works out to 0.063μL per mL of final solution volume.
- Cover reaction vessel with tin foil and let stir for at least 6 hours.
- Pipet the reaction mixture into dialysis tubing, seal, and dialyze. Use a buffer of 0.2M NaCl, pH 2.0, with at least 3 washings.
- Remove the reaction mixture from the tubing, then run on HPLC to purify and separate fragments.
- Cleavage occurs after a methionine residue, resulting in the conversion of methionine to homoserine lactone on the N-terminal fragment (causing a mass shift of -30).
- Ubiquitin mutants containing cysteine may require additional protection steps.
- Cyanogen Bromide is dangerous. All solutions containing cyanogen bromide (including used dialysis buffer), as well as exposed disposable vessels, should be disposed of appropriately.