| Assay Method Information | |
| | Fluorescence Polarization (FP) Competition Binding Assay |
| Description: | To establish assay conditions for competition binding, an enzyme titration/saturation binding experiment was initially performed. Bocillin-FL was prepared at 0.2 μM in a buffer comprised of 50 mM Hepes (pH 8.0), 300 mM NaCl and 5% (v/v) glycerol. Saturation binding was performed by mixing 40 μl of PBP solutions ranging in concentrations from 0-12 μM with 40 μl of the 0.2 μM Bocillin-FL solution, in individual wells of a black 384-well microplate. FP was measured immediately upon mixing (Excitation, 490 nm; Emission, 520 nm; g-factor, 0.96), using a Cytation3 (BioTek) microplate reader and measured continuously for up to 120 minutes. The FP response became stable after 30 minutes (80 minutes for PBP2), and showed a dose dependence on PBP concentration. For all PBPs the FP signal approached saturation with 1.5 μM PBP (final concentration). The competition binding assay was validated using beta-lactams ampicillin, aztreonam, mecillinam or meropenem. Assays (80 μl final volume) were performed with PBPs at a final concentration of 1.5 μM, Bocillin-FL at 0.1 μM and beta-lactam concentrations that ranged from 0-1000 μM. PBP3 was incubated with increasing concentrations of ampicillin or aztreonam in a black 384-well microplate (Corning) for 30 minutes, and PBP2 and PBP4 were likewise incubated with increasing concentrations of mecillinam and meropenem, respectively. Bocillin-FL was added and the FP immediately measured for 60 minutes (90 minutes for PBP2). |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |