Diagonal Electrophoresis for Detecting Disulfide Bridges

  • Alastair Aitken
  • Michèle Learmonth
Part of the Springer Protocols Handbooks book series (SPH)


Methods for identifying disulfide bridges have routinely employed “diagonal” procedures using two-dimensional paper or thin-layer electrophoresis. This essentially utilizes the difference in electrophoretic mobility of peptides containing either cysteine or cystine in a disulfide link, before and after oxidation with performic acid. It was first described by Brown and Hartley (1). Peptides unaltered by the performic acid oxidation have the same mobility in both dimensions and, therefore, lie on a diagonal. After oxidation, peptides that contain cysteine or were previously covalently linked produce one or two spots off the diagonal, respectively. This method has also been adapted for HPLC methodology and is discussed in Chapter 80.


Cyanogen Bromide Electrophoresis Buffer Paper Electrophoresis Xylene Cyanol Performic Acid 
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  1. 1.
    Brown, J. R., and Hartley, B. S. (1966) Location of disulphide bridges by diagonal paper electrophoresis Biochem. J. 101, 214–228.PubMedGoogle Scholar
  2. 2.
    Michl, H. (1951) Paper electrophoresis at potential differences of 50 volts per centimetre. Monatschr. Chem. 82, 489–493.CrossRefGoogle Scholar
  3. 3.
    Creighton, T. E. (1983) Disulphide bonds between cysteine residues, in Protein Structure—a Practical Approach (Rickwood, D. and Hames B. D., eds.), IRL, Oxford, UK, pp. 155–167.Google Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 1996

Authors and Affiliations

  • Alastair Aitken
    • 1
  • Michèle Learmonth
    • 1
  1. 1.National Institute for Medical ResearchMill Hill, LondonUK

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