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Nondenaturing Polyacrylamide Gel Electrophoresis of Proteins

  • John M. Walker
Part of the Springer Protocols Handbooks book series (SPH)

Abstract

SDS-PAGE (Chapter 21) is probably the most commonly used gel electro-phoretic system for analyzing proteins. However, it should be stressed that this method separates denatured protein. Sometimes one needs to analyze native, nondenatured proteins, particularly if wanting to identify a protein in the gel by its biological activity (for example, enzyme activity, receptor binding, antibody binding, and so on). On such occasions it is necessary to use a nondenatur-ing system such as described in this chapter. For example, when purifying an enzyme, a single major band on a gel would suggest a pure enzyme. However this band could still be a contaminant; the enzyme could be present as a weaker (even nonstaining) band on the same gel. Only by showing that the major band had enzyme activity would you be convinced that this band corresponded to your enzyme. The method described here is based on the gel system first described by Davis (1). To enhance resolution a stacking gel can be included (see  Chapter 21 for the theory behind the stacking gel system).

Keywords

Bromophenol Blue Ammonium Persulfate Refractive Index Change Electrophoresis Buffer Acrylamide Solution 
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

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Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • John M. Walker
    • 1
  1. 1.School of Life SciencesUniversity of HertfordshireHatfieldUK

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