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Assay Method Information |
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| | PHGDH Inhibition Assay |
| Description: | PHGDH assay buffer contained 50 mM TEA pH 8.0, 10 mM MgCl2, 0.05% BSA, and 0.01% Tween-20. PHGDH enzyme buffer consisted of assay buffer with 20 nM PHGDH and 0.2 mg/mL diaphorase. PHGDH substrate buffer contained 0.3 mM NAD+, 1.25 mM glutamate, 0.1 mM 3-phosphoglycerate, 0.2 mM resazurin, 1 μM PSAT1, and 1 μM PSPH. qHTS was performed in 1,536-well plates dispensed with a BioRAPTR FRD. Each well contained equal volumes of substrate buffer and assay buffer. Plates were read at 0 min and 20 min at room temperature with a ViewLux uHTS Microplate Imager (PerkinElmer). Follow-up assays were performed in black 384-well plates (Greiner) in 20 μL of enzyme buffer to which compounds were added in dose-response with an HP D300 digital dispenser (Hewlett-Packard), followed by addition of 20 μL of substrate buffer. Plates were incubated at room temperature (25 °C) and read at 0 and 20 min with a Spectramax M5 plate reader (Molecular Devices) in fluorescence intensity mode with |
| Affinity data for this assay | |
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