| Assay Method Information | |
| | Inhibition of Beta-Lactamase Enzymes |
| Description: | A buffer consisting of 0.1 M sodium phosphate (pH 7.0), 10 mM NaHCO3, and 0.005% Triton X-100 was used for all enzymes. The chromogenic substrate nitrocefin (SynGene, Bangalore, India) was used at 100 μM. Enzyme activity was monitored by the 490 nm absorbance increase upon nitrocefin hydrolysis. Assays were performed in clear polystyrene 384-well plates (Greiner Bio-One, Monroe, N.C.). Absorbance was measured for 1 hour at 30-s intervals using a Spectramax absorbance plate reader (Molecular Devices, Sunnyvale, Calif.). Measurement of beta-lactamase inhibition by INHIBITOR employed serial 3-fold dilutions of the inhibitor in assay buffer, ranging from 100 μM to 62.7 pM. A background absorbance progress curve for a control lacking enzyme and inhibitor was subtracted from each progress curve. |
| Affinity data for this assay | |
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