1. Analysis of whole cell lysates by Western blotting and 2D gel electrophoresis.A, cells overexpressing GFP fusion proteins and control cells were cultured as described under “Experimental Procedures.” Proteins of whole cell lysates were separated by means of SDS-PAGE and subsequently subjected to Western blotting analysis with antibodies to DnaK, GroEL, IbpA/B, Ffh, FtsY, SecA, and SecB.
Analysis of whole cell lysates by Western blotting and 2D gel electrophoresis.A, cells overexpressing GFP fusion proteins and control cells were cultured as described under “Experimental Procedures.” Proteins of whole cell lysates were separated by means of SDS-PAGE and subsequently subjected to Western blotting analysis with antibodies to DnaK, GroEL, IbpA/B, Ffh, FtsY, SecA, and SecB. B, comparative 2D gel analysis of proteins in whole cell lysates of cells overexpressing YidC-GFP and the control. 1.0 A600 unit of cells was solubilized, and proteins were separated by 2D gel electrophoresis. Proteins were visualized by silver stain, and differences between the replicate groups were analyzed using the PDQuest software (Bio-Rad). Each replicate group contained four independent samples. Differential protein accumulation was analyzed using the Student's t test and a 95% level of confidence (see “Experimental Procedures”). The indicated proteins were identified by MS from spots excised from gels stained with Coomassie Brilliant Blue R-250 or MS-compatible silver stain (Fig. 4 and Supplemental Table 1). Annotated spots were matched onto the silver-stained gels shown here using the PDQuest software (Bio-Rad). Numbers representing secretory proteins are in italic and those of chaperones in bold. The accumulation levels of spots numbered on the control gel were decreased, whereas the accumulation levels of spots numbered on the YidC-GFP overexpression gel were increased compared with the control.
Samuel Wagner et al. Mol Cell Proteomics 2007;6:
© 2007 The American Society for Biochemistry and Molecular Biology