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Two-dimensional gel electrophoresis (2-DE)

Part 3. Proteomics

3.2. Expression Proteomics

Basic Techniques and Approaches

  1. Emma McGregor1,
  2. Michael J. Dunn2

Published Online: 15 JAN 2005

DOI: 10.1002/047001153X.g302406

Encyclopedia of Genetics, Genomics, Proteomics and Bioinformatics

Encyclopedia of Genetics, Genomics, Proteomics and Bioinformatics

How to Cite

McGregor, E. and Dunn, M. J. 2005. Two-dimensional gel electrophoresis (2-DE). Encyclopedia of Genetics, Genomics, Proteomics and Bioinformatics.

Author Information

  1. 1

    Proteome Sciences plc, London, UK

  2. 2

    Proteome Research Centre, University College Dublin, Conway Institute for Biomolecular and Biomedical Research, Dublin, Ireland

Publication History

  1. Published Online: 15 JAN 2005

Abstract

Two-dimensional gel electrophoresis (2-DE) with immobilized pH gradients (IPGs) combined with protein identification by mass spectrometry (MS) is currently the method of choice in the majority of proteomic projects. In spite of the development of novel gel-free technologies, 2-DE remains the only technique that can be routinely applied to parallel quantitative expression profiling of large sets of complex protein mixtures such as whole-cell lysates. 2-DE involves the separation of solubilized proteins in the first dimension according to their charge properties (isoelectric point, pI) by isoelectric focusing (IEF) under denaturing conditions, followed by their separation in the second dimension by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), according to their relative molecular mass (Mr). As the charge and mass properties of proteins are essentially independent parameters, this orthogonal combination of charge (pI) and size (Mr) separations results in the sample proteins being distributed across the two-dimensional gel profile. 2-DE can resolve more than 5000 proteins simultaneously (∼2000 proteins routinely), and can detect <1 ng of protein per spot. Furthermore, it delivers a map of intact proteins, which reflects changes in protein expression level, isoforms, or posttranslational modifications. In this article, we outline the various steps in the 2-DE proteomics workflow, namely, sample preparation, solubilization, first-dimension IEF with IPGs, second-dimension SDS-PAGE, protein detection and visualization, and protein identification by mass spectrometry. The various parameters that must be considered and optimized for successful 2-DE separations of various types of sample are discussed.

Keywords:

  • immobilized pH gradient;
  • mass spectrometry;
  • protein detection;
  • protein identification;
  • proteomics;
  • sample preparation;
  • sample solubilization;
  • two-dimensional gel electrophoresis