Culture samples were grown under aerobic conditions to OD600 = 0.5. The cell pellets were re-suspended in SDS loading buffer (60 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol, 0.005% bromophenol blue, 5 mM EDTA, 0.1 M DTT) to a final concentration of 0.01 OD600 unit/µl and boiled for 5 min.
For detection of proteins a total of 0.05 OD600 unit of whole cell protein was loaded onto 4-12% Invitrogen NuPage (Novex) Bis-Tris mini gels. The gels were blotted for 60 min at 3.2 mA/cm2 in a Hoefer SemiPhor blotter tank (GE Healthcare) onto a PVDF membrane (Millipore) in transfer buffer (48 mM Tris pH 9, 20% methanol, 39 mM glycine, 0.0375% SDS). The α-GFP (Roche), α-GroEL (Sigma), α-FLAG (Sigma) monoclonal antibodies were diluted 1∶10.000, 1∶50.000 and 1∶10.000, respectively. The α-LeoA, α-TolC, α-Lep, α-OmpA, α-β-lactamase polyclonal antibodies were diluted 1∶1000, 1∶50, 1∶100 1∶3000 and 1∶3000, respectively.
Mouse and rabbit HRP conjugated secondary antibody was diluted 1∶2000 (Dako Cytomation). Blots were developed using Western Lightning Reagent (Perkin Elmer). The signal was detected and quantified using a ChemiDoc XRS station (BioRad).
For detection of proteins a total of 0.05 OD600 unit of whole cell protein was loaded onto 4-12% Invitrogen NuPage (Novex) Bis-Tris mini gels. The gels were blotted for 60 min at 3.2 mA/cm2 in a Hoefer SemiPhor blotter tank (GE Healthcare) onto a PVDF membrane (Millipore) in transfer buffer (48 mM Tris pH 9, 20% methanol, 39 mM glycine, 0.0375% SDS). The α-GFP (Roche), α-GroEL (Sigma), α-FLAG (Sigma) monoclonal antibodies were diluted 1∶10.000, 1∶50.000 and 1∶10.000, respectively. The α-LeoA, α-TolC, α-Lep, α-OmpA, α-β-lactamase polyclonal antibodies were diluted 1∶1000, 1∶50, 1∶100 1∶3000 and 1∶3000, respectively.
Mouse and rabbit HRP conjugated secondary antibody was diluted 1∶2000 (Dako Cytomation). Blots were developed using Western Lightning Reagent (Perkin Elmer). The signal was detected and quantified using a ChemiDoc XRS station (BioRad).