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Electroelution of Proteins from Polyacrylamide Gels

  • Paul Jenö
  • Martin Horst
Protocol
Part of the Springer Protocols Handbooks book series (SPH)

Abstract

Understanding the function of proteins requires determination of their structures. Advances in chemical technology make it possible to obtain a picture of global protein expression in model organisms such as the yeast Saccharomyces cerevisiae. Instrumental to these breakthroughs was the development of high-resolution two-dimensional gel electrophoresis and mass spectrometric means for rapid detection and analysis of peptides and proteins. While sequence information on single spots separated by two-dimensional electrophoresis can be quickly obtained by today’s technology, structural characterization of proteins expressed in low abundance is still a difficult task. For example, in our attempts to isolate components of the import machinery of yeast mitochondria, some proteins turned out to be expressed at extremely low levels, and it soon became evident that conventional purification techniques were impractical to obtain these proteins in amounts sufficient for structural analysis. Furthermore, some proteins of the mitochondrial import machinery were rapidly degraded by proteases, and they could be obtained intact only by first denaturing mitochondria with trichloroacetic acid and then solubilizing them in boiling sample buffer for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (1). This in turn made the separation of several milligrams of denatured proteins necessary, which was best achieved by preparative SDS-PAGE followed by electroelution of individual proteins.

Keywords

Hydrophilic Interaction Chromatography Import Machinery Ammonium Hydrogen Carbonate Electrophoresis Chamber Global Protein Expression 
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

  • Paul Jenö
    • 1
  • Martin Horst
    • 2
  1. 1.Department of BiochemistryBiozentrum der Universität BaselSwitzerland
  2. 2.STRATEC MedicalOberdorfSwitzerland

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