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Comparative Analysis of Cytoplasmic Membrane Proteomes of Escherichia coli Using 2D Blue Native/SDS-PAGE

  • Susan Schlegel
  • Mirjam Klepsch
  • David Wickström
  • Samuel Wagner
  • Jan-Willem de Gier
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 619)

Abstract

Two-dimensional blue native (2D BN)/SDS-PAGE is the method of choice for the global analysis of the subunits of complexes in membrane proteomes. In the 1st dimension complexes are separated by BN-PAGE, and in the 2nd dimension their subunits are resolved by SDS-PAGE. The currently available protocols result in the distortion of the 1st dimension BN-gel lanes during their transfer to the 2nd dimension separation gels. This leads to low reproducibility and high variation of 2D BN/SDS-gels, making 2D BN/SDS-PAGE unsuitable for comparative analysis. Here, we present a 2D BN/SDS-PAGE protocol where the 1st dimension BN-gel is cast on a GelBond PAG film. Immobilization prevents distortion of BN-gel lanes when they are transferred to the 2nd dimension, which lowers variation and greatly improves reproducibility of 2D BN/SDS-gels. The use of 2D BN/SDS-PAGE with an immobilized first dimension is illustrated by the characterization of the cytoplasmic membrane proteome of Escherichia coli cells overexpressing cytochrome bo 3.

Key words

Membrane protein membrane isolation protein complex two-dimensional blue native SDS-PAGE comparative proteomics E. coli 

Notes

Acknowledgements

This research was supported by grants from the Swedish Research Council and The Center for Biomembrane Research, which is supported by the Swedish Foundation for Strategic Research.

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Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Susan Schlegel
    • 1
  • Mirjam Klepsch
    • 1
  • David Wickström
    • 1
  • Samuel Wagner
    • 1
  • Jan-Willem de Gier
    • 1
  1. 1.Center for Biomembrane Research, Department of Biochemistry and BiophysicsStockholm UniversityStockholmSweden

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