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Protein Solubility in Two-Dimensional Electrophoresis

Basic Principles and Issues

  • Protocol
The Protein Protocols Handbook

Part of the book series: Springer Protocols Handbooks ((SPH))

Abstract

The solubilization process for two-dimensional (2-D) electrophoresis has to achieve four parallel goals: Breaking macromolecular interactions in order to yield separate polypeptide chains. This includes denaturing the proteins to break noncovalent interactions, breaking disulfide bonds, and disrupting noncovalent interactions between proteins and nonproteinaceous compounds such as lipids or nucleic acids. Preventing any artefactual modification of the polypeptides in the solubilization medium. Ideally, the perfect solubilization medium should freeze all the extracted polypeptides in their exact state prior to solubilization, both in terms of amino acid composition and in terms of posttranslational modifications. This means that all the enzymes able to modify the proteins must be quickly and irreversibly inactivated. Such enzymes include of course proteases, which are the most difficult to inactivate, but also phosphatases, glycosidases, and so forth. In parallel, the solubilization protocol should not expose the polypeptides to conditions in which chemical modifications (e.g., deamidation of Asn and Gln, cleavage of Asp-Pro bonds) may occur.

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Authors and Affiliations

  1. iRTSV/LBBSI, CEA Grenoble, Grenoble, France

    Thierry Rabilloud

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  1. Thierry Rabilloud
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Editors and Affiliations

  1. School of Life Sciences, University of Hertfordshire, Hatfield, Hertfordshire, UK

    John M. Walker

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© 2009 Humana Press, a part of Springer Science+Business Media, LLC

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Rabilloud, T. (2009). Protein Solubility in Two-Dimensional Electrophoresis. In: Walker, J.M. (eds) The Protein Protocols Handbook. Springer Protocols Handbooks. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-59745-198-7_11

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  • DOI: https://doi.org/10.1007/978-1-59745-198-7_11

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-60327-474-6

  • Online ISBN: 978-1-59745-198-7

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