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Two-Dimensional PAGE Using Immobilized pH Gradients

  • Graeme R. Guy
  • Robin J. Philp
Protocol
Part of the Springer Protocols Handbooks book series (SPH)

Abstract

Since its introduction some 20 years ago by O’Farrell (1), two-dimensional polyacrylamide gel electrophoresis (2D PAGE) has remained an extremely powerful method for the separation of complex protein mixtures, such as whole-cell lysates. In the first dimension, proteins are separated by charge using isoelectric focusing (IEF) and, in the second, by size employing sodium dodecyl sulfate (SDS)-PAGE. The combination of these two techniques results in a highly resolving separation that can be used for many applications, such as gene regulation studies, comparison of protein phosphorylation, and protein purification. At higher loadings, identification of individual proteins can be carried out using such methods as microsequencing or mass spectrometry.

Keywords

Sodium Dodecyl Sulfate Carrier Ampholyte Complex Protein Mixture Gene Regulation Study Plastic Backing 
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.

References

  1. 1.
    O’Farrell, P. H. (1975) High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250, 4007–4021.PubMedGoogle Scholar
  2. 2.
    Gorg, A. (1992) Two-dimensional electrophoresis. Nature 349, 545,546.CrossRefGoogle Scholar
  3. 3.
    Hanash, S. M., Strahler, J. R., Neel, J. V., Hailat, N., Melhem, R., Keim, D., Zhu, X. X., Wagner, D., Gage, D. A., and Watson, J. T. (1991) Proc. Natl. Acad. Sci. USA 88, 5709–5713.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 1996

Authors and Affiliations

  • Graeme R. Guy
    • 1
  • Robin J. Philp
    • 1
  1. 1.Institute of Molecular and Cell BiologyNational University of SingaporeSingapore

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