2D BN-PAGE methodology suitable for relative quantification and 2D BN-PAGE reference map of the E. coli cytoplasmic membrane proteome.A, to be able to use 2D BN-PAGE for relative quantification, a 1.0-mm-thick first dimension gel was cast on a polyester support (GelBond PAG film, Cambrex) (step 1). 2D BN-PAGE methodology suitable for relative quantification and 2D BN-PAGE reference map of the E. coli cytoplasmic membrane proteome.A, to be able to use 2D BN-PAGE for relative quantification, a 1.0-mm-thick first dimension gel was cast on a polyester support (GelBond PAG film, Cambrex) (step 1). After the first dimension blue native run, lanes were cut (step 2); equilibrated in denaturing, reducing, and alkylating buffer; and submerged into a low melting agarose solution on top of a 1.5-mm-thick 10% Duracryl gel (step 3). 6–12 gels were run in parallel in the second dimension in an Ettan DALTtwelve system (GE Healthcare) (step 4). Relative quantification of spot intensities was performed using PDQuest from Bio-Rad (step 5). B, the cytoplasmic membrane proteome of E. coli BL21(DE3)pLysS was resolved by 2D BN-PAGE. 150 μg of cytoplasmic membrane protein were used for the analysis. Proteins were visualized by colloidal Coomassie stain. Proteins in indicated spots were identified by MS (Table II and Supplemental Table 3). Samuel Wagner et al. Mol Cell Proteomics 2007;6:1527-1550 © 2007 The American Society for Biochemistry and Molecular Biology