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 ofEscherichia colicells overexpressing cytochromebo3 .
