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.
Free full text: Click here
