Cyanogen Bromide Cleavage of Ubiquitin
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.