title: Comparative analysis of cytoplasmic membrane proteomes of Escherichia coli using 2D blue native/SDS-PAGE.
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).
authors.affiliation: Center for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden.
authors.forename: Susan
authors.initials: S
authors.lastname: Schlegel
authors.forename: Mirjam
authors.initials: M
authors.lastname: Klepsch
authors.forename: David
authors.initials: D
authors.lastname: Wickström
authors.forename: Samuel
authors.initials: S
authors.lastname: Wagner
authors.forename: Jan-Willem
authors.initials: JW
authors.lastname: de Gier
mesh_terms: D002462:Cell Membrane / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D020543:Proteome / Q000378:metabolism*
chemical_list: D020543:Proteome