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CNBR Digest of Protein

(based on method in "A Practical Guide to Protein and Peptide Purification for Microsequencing", 2nd Ed.,Paul Matsudaira ,Editor, Academic Press, N.Y., 1993)

  1. Suspend protein in 70% formic acid. (note: Formic acid is usually obtained as an 88% solution). We have used up to about 100mg protein per 100 ml of formic acid.
  2. Make up a 70 mg/ml or 0.66M CnBr stock solution: 132 ml of 5M CnBr in acetonitrile and 868 ml of 70% formic acid. CnBr is very dangerous and should be handled accordingly.
  3. Add 10 ml of the 70 mg/ml CnBr solution per 10 mg of protein.
  4. Incubate in the dark at room temperature for 24 hours.
  5. Speed-Vac the the tube contents to dryness. Add an additional 100 ml of water to the tube and re-dry in the Speed-Vac.
  6. The protein should now be cleaved at Met residues, and the resulting peptides may be separated on HPLC or by PAGE depending on the size and amounts of the peptides.