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Protein Solubility in Two-Dimensional Electrophoresis

Basic Principles and Issues
  • Thierry Rabilloud

Abstract

The solubilization process for two-dimensional (2-D) electrophoresis needs to achieve four parallel goals:
  1. 1.

    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.

     
  2. 2.

    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.

     
  3. 3.

    Allowing the easy removal of substances that may interfere with 2-D electrophoresis. In 2-D electrophoresis, proteins are the analytes. Thus, anything in the cell but proteins can be considered as an interfering substance. Some cellular compounds (e.g., coenzymes, hormones) are so dilute they go unnoticed. Other compounds (e.g., simple nonreducing sugars) do not interact with proteins or do not interfere with the electrophoretic process. However, many compounds bind to proteins and/or interfere with 2-D electrophoresis and must be eliminated prior to electrophoresis if their amount exceeds a critical interference threshold. Such compounds mainly include salts, lipids, polysaccharides (including cell walls), and nucleic acids.

     
  4. 4.
    Keeping proteins in solution during the 2-D electrophoresis process. Although solubilization stricto sensu stops at the point where the sample is loaded onto the first dimension gel, its scope can be extended to the 2-D process per se, as proteins must be kept soluble until the end of the second dimension. Generally speaking, the second dimension is a sodium dodecyl sulfate (SDS) gel, and very few problems are encountered once the proteins have entered the SDS-polyacrylamide gel electrophoresis (SDS-PAGE) gel. The one main problem is overloading of the major proteins when micropreparative 2-D electrophoresis is carried out, and nothing but scaling-up the SDS gel (its thickness and its other dimensions) can counteract overloading a SDS gel. However, severe problems can be encountered in the isoelectric fusing (IEF) step. They arise from the fact that IEF must be carried out in low ionic strength conditions and with no manipulation of the polypeptide charge. IEF conditions give problems at three stages:
    1. a.

      During the initial solubilization of the sample, important interactions between proteins of widely different pI and/or between proteins and interfering compounds (e.g., nucleic acids) may occur. This yields poor solubilization of some components.

       
    2. b.

      During the entry of the sample in the focusing gel, there is a stacking effect due to the transition between a liquid phase and a gel phase with a higher friction coefficient. This stacking increases the concentration of proteins and may give rise to precipitation events.

       
    3. c.

      At, or very close to, the isoelectric point, the solubility of the proteins comes to a minimum. This can be explained by the fact that the net charge comes close to zero, with a concomitant reduction of the electrostatic repulsion between polypeptides. This can also result in protein precipitation or adsorption to the IEF matrix.

       
     

Keywords

Sodium Dodecyl Sulfate Noncovalent Interaction Alkyl Tail Zwitterionic Detergent Sample Entry 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Humana Press Inc., Totowa, NJ 2002

Authors and Affiliations

  • Thierry Rabilloud
    • 1
  1. 1.DBMS/BECP, CEA-GrenobleGrenobleFrance

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