Relative quantification of the cytoplasmic membrane proteome by 2D BN-PAGE. Relative quantification of the cytoplasmic membrane proteome by 2D BN-PAGE. Cytoplasmic membranes of cells overexpressing GFP fusion proteins and of control cells were isolated as described under “Experimental Procedures.” Independent triplicates of cytoplasmic membrane isolates were analyzed by 2D BN-PAGE. Proteins were visualized by colloidal Coomassie staining, and differences between replicate groups were analyzed using PDQuest (Bio-Rad). Differential protein expression was analyzed using the Student's t test with a 95% level of confidence and a threshold for quantitative differences of at least 2-fold upon membrane protein overexpression (see “Experimental Procedures”). Proteins were identified by MS (Fig. 10 and Supplemental Table 3). Identified proteins with differential accumulation levels are indicated by gene names. The accumulation levels of spots indicated on the control gel were decreased, whereas the levels of spots indicated on the YidC-GFP overexpression gel were increased compared with the control. LacY is in parentheses because its levels were significantly increased upon GST-GFP overexpression, however, not upon membrane protein overexpression. A slight reduction of purity (85% instead of 92% as calculated by densitometry) of the cytoplasmic membranes isolated from overexpressors was obvious from the stronger spots for OmpA, OmpF, and the recently identified outer membrane assembly complex (YaeT, YfgL, NlpB, and YfiO) in our 2D BN-PAGE gels (75). Samuel Wagner et al. Mol Cell Proteomics 2007;6:1527-1550 © 2007 The American Society for Biochemistry and Molecular Biology