Streptavidin peroxidase conjugate

Western ligand affinity blotting was performed as previously described [4 (link)], with modifications. Briefly, overnight cultures were diluted 1:50 in TSB medium and grown at 37°C with shaking for 4 hours. Cells were washed and surface-associated protein extracts were prepared through resuspension in PBS media containing 20 μg/mL lysostaphin (Sigma), 20 μg/mL DNAse (New England Biolabs), 1 mM phenylmethanesulfonylfluoride (PMSF, Thermo Scientific), and 1:100 dilution of a protease inhibitor cocktail (Sigma, P2714). Cell extracts were incubated at 37°C for 30 minutes and spun at 12,000 x g for 1 minute at 4°C. The protein concentration in the supernatant was determined with a BCA Protein Assay Kit (Pierce), and 20 μg of each sample was separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). FnBPs were detected by incubation for 1 hour with biotinylated human fibronectin (50 μg/mL) in PBS containing 0.1% Tween 20 (PBST). An EZ-Link sulfo-N-hydroxysuccinimide-LC biotinylation kit (Pierce) was used to biotinylate human fibronectin (Sigma). After washing with PBST, membranes were incubated for 1 hour with streptavidin-peroxidase conjugate (Roche; 1:3000 dilution). Finally, membranes were developed with the ECL Western blotting system (Pierce). A S. aureus fnbA fnbB double mutant [19 (link)] was used as a negative control.

The reverse line blot assay was carried out according to Kong et al.[6 (link)]. Briefly, a Biodyne C membrane was activated with 16% (w/v) 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) for 20 min and rinsed with sterile milliQ water. The membrane was placed in a Miniblotter® (Immunetics) and specific 5’ amine labeled oligonucleotide probes were then bound to the membrane and fixed with 0.1 M NaOH. The membrane was then removed, rotated 90° and placed back into the Miniblotter. Products from the five mPCR were combined and boiled for 5 min before being applied to the membrane perpendicular to the bound probes. The membrane was then incubated at 55°C for 1 hr before washing in 1× SSPE (150 mM sodium chloride, 10 mM sodium phosphate and 1 mM EDTA)/0.1% SDS solution. A streptavidin-peroxidase conjugate (Roche) was then added and the membrane further incubated at 42°C for 1 hr before washing and exposing with enhanced chemiluminescence (ECL) detection kit (GE LifeSciences). Chemiluminescence was detected using LAS3000 Imager (Fujifilm).
A clear hybridisation signal was interpreted as the presence of the corresponding gene, whereas the lack of signal was interpreted as the absence of a gene. Faint or indistinct hybridisations deemed ambiguous were confirmed using individual PCR and agarose gel electrophoresis.