PBP2a enzymatic activity inhibition assay

The catalytic activity of PBP2a can be monitored at 482 nm using nitrocefin, a colorimetric substrate⁽²⁴⁾. 10 μM BLIP-II was incubated with 2 μM PBP2a in 25 mM HEPES, 1.0 M NaCl for 45 minutes at room temperature before nitrocefin was added. The hydrolysis of nitrocefin was monitored in 20 second time intervals at 37°C in a 96-well plate format using a Tecan Infinite Pro 200 plate reader.