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Mitochondria pp 315-324 | Cite as

Two-Dimensional Blue Native/Blue Native Polyacrylamide Gel Electrophoresis for the Characterization of Mitochondrial Protein Complexes and Supercomplexes

  • Stephanie Sunderhaus
  • Holger Eubel
  • Hans-Peter Braun
Part of the Methods in Molecular Biology™ book series (MIMB, volume 372)

Abstract

Blue native polyacrylamide gel electrophoresis (BN-PAGE) employs the dye Coomassie for the labeling of proteins and protein complexes under native conditions. Electrophoresis under native conditions subsequently allows resolution of proteins and protein complexes according to their molecular mass. BN-PAGE can be combined with second gel dimensions. Best known is the two-dimensional (2D)-BN/sodium dodecyl sulfate (SDS)-PAGE system, which allows resolution of subunits of protein complexes. A 2D-BN/BN-PAGE system was developed that proved useful for investigating the substructure of protein complexes and protein supercomplexes. The basis of this 2D system is a variation of the conditions used for the two BN gel dimensions. Here, we present a basic protocol for the analysis of mitochondrial fractions by 2D-BN/BN-PAGE. Because both gel dimensions are carried out under native conditions, the 2D-BN/BN system is compatible with in-gel enzyme activity staining.

Key Words

Blue native polyacrylamide gel electrophoresis (BN-PAGE) digitonin in-gel enzyme assay membrane proteins protein complexes protein supercomplexes respiratory chain 

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Copyright information

© Humana Press Inc., Totowa, NJ 2007

Authors and Affiliations

  • Stephanie Sunderhaus
    • 1
  • Holger Eubel
    • 1
  • Hans-Peter Braun
    • 1
  1. 1.Abt. Angewandte Genetik, Naturwissenschaftliche FakultätUniversität HannoverHannoverGermany

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