4.5. Bocillin-FL Binding Assay

The β-lactam antibiotics affinity of PBP4^TO was tested using a fluorescent penicillin derivative Bocillin-FL (Thermo-Fisher) [44]. One microgram of PBP4^TO in 10 mM Tris-HCl buffer (pH 7.5) with 50 mM NaCl and 10 mM MgCl₂ was incubated with a gradient of antibiotics at 28 °C for 20 min. Then, 15 μg/mL Bocillin-FL was added to the reaction mixture and further incubated at 28 °C for 30 min. The reactions were stopped by addition of 5x SDS-PAGE sample buffer. Samples were loaded onto a 12% SDS-PAGE gel and proteins were separated by electrophoresis for 2 h at 120 V. The Bocillin-FL signal was detected with an Odyssey Fc imaging system (LI-COR Biosciences) at 600 nm.