title: Immobilization of the first dimension in 2D blue native/SDS-PAGE allows the relative quantification of membrane proteomes.
abstract: In biological membranes many proteins are organized in complexes. The method of choice for the global analysis of the subunits of these complexes is two-dimensional blue native (2D BN)/SDS-PAGE. In the 1st dimension complexes are separated by BN-PAGE, and in the 2nd dimension their subunits are resolved by SDS-PAGE. In the currently available protocols the 1st dimension BN gel lanes get distorted during their transfer to the 2nd dimension separation gels. This leads to low reproducibility and high variation of 2D BN/SDS-gels, rendering them unsuitable for comparative analysis. We have developed 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, which lowers variation and greatly improves reproducibility of 2D BN/SDS-gels. 2D BN/SDS-PAGE with an immobilized 1st dimension was used for the comparative analysis of the cytoplasmic membrane proteomes of Escherichia coli cells overexpressing a membrane protein and to create a 2D BN/SDS-PAGE reference map of the E. coli cytoplasmic membrane proteome with 143 identified proteins from 165 different protein spots.
authors.affiliation: Center for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University, SE-106 91 Stockholm, Sweden.
authors.forename: Mirjam
authors.initials: M
authors.lastname: Klepsch
authors.forename: Susan
authors.initials: S
authors.lastname: Schlegel
authors.forename: David
authors.initials: D
authors.lastname: Wickström
authors.forename: Giulia
authors.initials: G
authors.lastname: Friso
authors.forename: Klaas J
authors.initials: KJ
authors.lastname: van Wijk
authors.forename: Jan-Olov
authors.initials: JO
authors.lastname: Persson
authors.forename: Jan-Willem
authors.initials: JW
authors.lastname: de Gier
authors.forename: Samuel
authors.initials: S
authors.lastname: Wagner
mesh_terms: D015180:Electrophoresis, Gel, Two-Dimensional / Q000295:instrumentation / Q000379:methods*; D029968:Escherichia coli Proteins / Q000302:isolation & purification; D055767:Immobilized Proteins; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000032:analysis*; D020543:Proteome / Q000032:analysis; D040901:Proteomics / Q000379:methods*
chemical_list: D029968:Escherichia coli Proteins; D055767:Immobilized Proteins; D008565:Membrane Proteins; D020543:Proteome