Characterization of Protein Preparations by SDS-PAGE and Mass Spectrometry

Purity of FliC preparations was assessed by SDS-PAGE, Coomassie staining and western blots. Protein samples (between 1.0 and 12.5 μg per lane, unless otherwise stated and depending on the assay), representing whole cell lysates or flagella extract, were separated in 12% SDS-PAGE gels run at 200 V for 40 min. For Coomassie staining, gels were stained with brilliant blue solution for 1 h and de-stained (1% acetic acid) overnight for visualization using a Chemidoc XRS (Bio-Rad). For western blot, the gels were transferred to 0.45 μm nitrocellulose membranes (Bio-Rad #162-0115, USA) for 1 h at 100 V with cooling, and blocked with 5% skim milk in PBS-T. The membranes were incubated with a 1/100 dilution of polyclonal rabbit anti-flagella H-pool-E (H48+others) or pool-A (H1+others) antibody (Staten Serum Institut, Denmark) for 1 h and washed three times in PBS-T for 5 min. The secondary antibody was a 1:500 dilution of goat anti-rabbit IgG HRP-conjugate (Santa Cruz Biotech #sc-2030, USA) or Goat anti-rabbit IgG-AP (1:10,000; Santa Cruz Biotechnology) for 1 h, subsequently washed four times in PBS-T (5 min). Blots were developed using 3,3′-Diaminobenzidine substrate (Sigma #D4418, USA) or 5-bromo-4-chloro-3-indolyl phosphate (BCIP)/nitro blue tetrazolium (NBT) (Sigma). The reactions were stopped by addition of water prior to image capture using a flatbed scanner (Epson, Japan) or Chemidoc XRS.

Mass spectrophotometry (MS) was performed to identify several extraneous unknown proteins found to be present in initial FliC preparations. The MS was carried out using protein samples derived from CFT073Δ4 or its fliC-deficient derivative. Proteins (2.5 μg) were resolved by SDS-PAGE, stained with Coomassie blue and de-stained (1% acetic acid) overnight. Proteins bands were isolated in 1% acetic acid and were analyzed at the Translational Research Institute (University of Queensland), Proteomics Core Facility, Brisbane.