Typhoon 9500 fluorescence imager

Bocillin labeling of PBPs was performed as described previously (Cho et al., 2016 (link)) with the following modifications. After incubation with Bocillin washing the cell pellets 3X with PBS (137 mM NaCl, 2.7 mM KCl, 8 mM Na₂HPO₄, and 2 mM KH₂PO₄), the pellets were frozen at −80°C. Cell pellets were then thawed, resuspended in 1 mL of PBS and lysed using a FastPrep-24 (MPBio). Cells were lysed using matrix B in 2 mL tubes for 40 s at 6 m/s. Cells were centrifuged for 3 min at 7,000 rpm to remove the undisrupted cells and matrix and the supernatant was transferred to a fresh tube. Membranes were then pelleted by ultracentrifugation at 100,000 × g for 20 min at 4°C. The membrane pellets were then washed with 1X PBS and resuspended in 60 μL 1X PBS. Resuspended samples were mixed with 60 μL 2X Laemmli sample buffer (100 mM Tris-Cl (pH 6.8), 4% (w/v) SDS, 0.2% (w/v) bromophenol blue, 20% (v/v) glycerol, 5% (v/v) β-mercaptoethanol) and boiled for 10 min at 95°C. After measuring the total protein concentration of each sample with the NI-protein assay (G-Biosciences), an equivalent amount of total protein for each sample was then separated on a 10% SDS-PAGE gels. Bocillin-labeled proteins were then imaged using a Typhoon 9500 fluorescence imager (GE Healthcare) with excitation at 488 nm and emission at 530 nm.

Bocillin-binding assays on membrane extracts were performed as described previously [8 (link)]. For bocillin binding assays of purified proteins, 8.3 μM of protein and 250 μM Bocillin-FL (ThermoFisher cat# B13233) were incubated for 30 minutes at room temperature. The protein was then combined with sample buffer and 10 pmol/lane was run on a 4–20% polyacrylamide gel. Bocillin gels were imaged using a Typhoon 9500 fluorescence imager (GE Healthcare) with excitation at 488 nm and emission at 530 nm.