Biofilm cell-wall associated protein preparation

Overnight cultures were diluted (1:100) into fresh TSBG, aliquoted into six-well (3.6 mL) tissue-culture treated polystyrene plates (Corning, #CLS3516), and incubated statically for at 37°C for 24 h. Supernatants were discarded, biofilms were washed two times with 3.6 mL sterile PBS, resuspended in 1 mL sterile PBS, normalized to OD₆₀₀, and centrifuged (12,000 x g for two min). Biofilm pellets were washed twice with PBS (1 mL), re-suspended in a 48 μL mixture of lysostaphin (20 μg/mL), 1x Halt Protease Inhibitor in TSM buffer (10 mM MgCl₂ 500 mM Sucrose in 50 mM Tris, pH7.5), and incubated for 30 min at 37°C. Samples were centrifuged (12,000 x g for two min) and the supernatant (36 μL) was mixed with 4x SDS (12 μL) sample buffer [200mM Tris-Cl (pH 6.8), 588mM beta-mercaptoethanol, 8% SDS, 0.08% bromophenol blue, 40% glycerol, 50mM EDTA]. Samples were boiled for 10 min and stored at −80C.