Two-Dimensional PAGE Using Flat-Bed IEF in the First Dimension

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


Two-dimensional polyacrylamide gel electrophoresis (2D PAGE) of proteins involves the use of two independent separation techniques resulting in the ability to resolve very complex mixtures. Normally, isoelectric focusing (IEF) and sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) are used in the first and second-dimensions, respectively (1). Traditionally, the first dimension, IEF, has been run by casting the gels in glass capillaries that are subsequently loaded into a tank vertically, with an upper and lower reservoir. Samples can be applied to the space above the gel, and separation occurs by applying an electric field across the two ends of the gel. After the required separation time, the gels are removed by extruding them from the capillaries using a water-filled syringe to displace them directly into a suitable equilibration buffer.


Equilibration Buffer Carrier Ampholyte Flat Horizontal Surface Plastic Backing Equilibration Tube 
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  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

Copyright information

© Humana Press Inc., Totowa, NJ 1996

Authors and Affiliations

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

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