Research Article

About thiol derivatization and resolution of basic proteins in two-dimensional electrophoresis

  1. Sylvie Luche1,
  2. Hélène Diemer2,
  3. Chistophe Tastet1,
  4. Mireille Chevallet1,
  5. Alain Van Dorsselaer2,
  6. Emmanuelle Leize-Wagner2,
  7. Thierry Rabilloud1,*

Article first published online: 22 JAN 2004

DOI: 10.1002/pmic.200300589

Issue

PROTEOMICS

PROTEOMICS

Volume 4, Issue 3, pages 551–561, March 2004

Additional Information

How to Cite

Luche, S., Diemer, H., Tastet, C., Chevallet, M., Van Dorsselaer, A., Leize-Wagner, E. and Rabilloud, T. (2004), About thiol derivatization and resolution of basic proteins in two-dimensional electrophoresis. PROTEOMICS, 4: 551–561. doi: 10.1002/pmic.200300589

Author Information

  1. 1

    CEA – Laboratoire de Bioénergétique Cellulaire et Pathologique, DRDC/BECP Grenoble, France

  2. 2

    Laboratoire de Spectrométrie de Masse Bio-Organique, UMR CNRS 7509, ECPM, Strasbourg, France

*DRDC/BECP, CEA-Grenoble, 17 rue des martyrs, F-38054 Grenoble Cedex 9, France Fax: +33-76-88-51-87

Publication History

  1. Issue published online: 19 FEB 2004
  2. Article first published online: 22 JAN 2004
  3. Manuscript Received: 10 JUN 2003

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Keywords:

  • Basic proteins;
  • Cysteine;
  • Maleimide;
  • Two-dimensional gel electrophoresis

Abstract

The influence of thiol blocking on the resolution of basic proteins by two-dimensional electrophoresis was investigated. Cysteine blocking greatly increased resolution and decreased streaking, especially in the basic region of the gels. Two strategies for cysteine blocking were found to be efficient: classical alkylation with maleimide derivatives and mixed disulfide exchange with an excess of a low molecular weight disulfide. The effect on resolution was significant enough to allow correct resolution of basic proteins with in-gel rehydration on wide gradients (e.g. 3–10 and 4–12), but anodic cup-loading was still required for basic gradients (e.g. 6–12 or 8–12). These results demonstrate that thiol-related problems are not solely responsible for streaking of basic proteins on two-dimensional gels.

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