Bovine fibronectin

Maxinunc microtiter plates (eBiosciences) were coated with 0.5 μg human collagen I (Corning), human collagen VI (Abcam), bovine fibronectin (Corning), mouse collagen IV (Corning) and mouse laminin (Corning) at 4°C for overnight, and blocked with 3% BSA for 1 h at 37 °C. Recombinant His-tagged BBA33 protein was serially diluted 1:2 starting from 4 μM down to 62.5 nM in PBS, 0.1% Tween-20, and added to coated wells in triplicate and incubated for 1 h at 37 °C. After five washes in PBS, 0.1% Tween-20, a 1:6000 dilution of monoclonal antibody directed against poly-His (His₆) was added to each well and incubated for 1 h at 37°C. Following five washes in PBS, 0.1% Tween-20, a 1:4000 dilution of HRP conjugated antimouse IgG was added to each well and incubated for 1 h at 37°C. The wells were then washed in PBS, 0.1% Tween-20, after which 3,3′,5,5′-tetramethylbenzidine was added as substrate. The enzymatic reaction was stopped after 3 min using 0.16 M sulfuric acid (Thermo scientific), and the absorbance at 450 nm was determined.