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Updated: Jun 13, 2026
Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli
Published on: January 6, 2015
Comparative analysis of cytoplasmic membrane proteomes of Escherichia coli using 2D blue native/SDS-PAGE
Susan Schlegel1, Mirjam Klepsch, David Wickström
1Center for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden.
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).
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