| Assay Method Information | |
| | PDF Inhibition Assay |
| Description: | Enzyme assay was carried out in 96-well microtiter plates with a SpectraMax plate reader (Molecular Devices Corp.). PDF activity was measured using a continuous enzyme-linked assay. The reaction was triggered with the addition of PDF enzyme, and absorbance was monitored for 15 min at 340 nm. For determination of Km values, substrate concentration was varied up to 45 mM. A standard curve with NADH concentrations ranging from 0 to 1 mM was performed. The velocities obtained in mOD340 nm per minute were then converted to micromolar NADH produced per second to calculate Vmax. To determine IC50s, PDF activity was measured in the presence of increasing concentrations of the inhibitor in the presence of substrate concentration corresponding to Km values. All data fitting was carried out with nonlinear least squares regression using the commercial software package Graphit. |
| Affinity data for this assay | |
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