Quantifying A. nosocomialis PilA Production

The production of PilA, which is a type IV pilin produced by A. nosocomialis (Harding et al., 2013 (link)) was determined by western blotting using anti-PilA polyclonal rabbit antisera generated as described (Harding et al., 2015 (link)). Briefly, cells of the ATCC 17978, NFAb-1 and NFAb-2 strains were collected from the plastic surface of motility plates after the agarose layer of MMA plates was removed, while ATCC 19606^T cells, which do not display twitching motility, were collected from the surface of MMA plates. Cells were resuspended in sterile PBS and OD₆₀₀ values were determined to normalize cell numbers prior to lysis. Cell pellets were then lysed in 100 µl of 5X SDS sample buffer (250 mM Tris-HCl, pH6.8; 10% SDS; 30% glycerol; 5% beta-mercaptoethanol and 0.02% bromophenol blue) and boiled for 5 minutes. Equal volumes of cell lysates were loaded onto 4-12% NuPAGE Bis-Tris gels (Invitrogen), transferred to Immobilon-PSQ PVDF membranes (Millipore). Membranes were incubated at 4°C with a 1:1,000 dilution of anti-PilA serum for 16 h. Immunocomplexes were detected by chemiluminescence using an 1:5,000 dilution of anti-rabbit IgG HRP-linked secondary antibody (GE Healthcare) and ECL Prime Western Blotting Detection Reagent (GE Healthcare).