Protein Protocols Protein Protocols

460 Aitken and Learmonth
4. Notes
1. Deionization of urea: Urea should be deionized immediately before use to remove
cyanates, which react with amino and thiol groups. The method is to filter the urea solution
through a mixed-bed Dowex or Amberlite resin in a filter flask.
2. The choice of buffer is dependent on the solubility of the protein. Distilled water may be
used or it may be necessary to use 0.1 M NaCl or 0.2 M sodium borate buffer in the
presence of 8 M deionized urea.
3. Other dicarboxylic anhydrides may be used. Particularly common is citraconic anhydride
(3). This reagent has the advantage of reversibility, being readily removed with acid. When
used in conjunction with trypsin, the proteolytic reaction may be stopped by reducing the
pH, which will also remove the lysine blocking groups.
References
1. Klapper, M. H. and Klotz, I. M. (1972) Meth. Enzymol. 25, 531–536.
2. Hollecker, M. and Creighton, T. E. (1980) Counting integral numbers of amino groups per
polypeptide chain: FEBS Lett. 119, 187–189.
3. Yarwood, A. (1989) Manual methods of protein sequencing, in Protein Sequencing-A
Practical Approach, (Findlay J. B. C. and Geisow, M. J., eds.) IRL Press, Oxford, pp.
119–145.