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| | LoxA HTP Assay |
| Description: | Briefly, 3 uL of enzyme (approximately 20 nM LoxA, final concentration) or buffer (no-enzyme control) was dispensed into 1536-well Greiner black clear-bottom assay plates using a BioRAPTR Flying Reagent Dispenser (Beckman Coulter, Fullerton, CA). Compounds and controls [broad spectrum LOX inhibitor, nordihydroguaiaretic acid (NDGA), concentration ranging from 3.4 mM to 0.208 uM] were transferred (23 or 46 nL) via Kalypsys PinTool, equipped with a 1536-pin array, to yield final library compound concentrations ranging from 114 uM to 0.73 nM. The plate was covered and incubated for 15 min at room temperature, followed by addition of a 1 uL aliquot of a substrate solution (40 uM arachidonic acid, final concentration; KM = 10 uM) to start the reaction, for a final assay volume of 4 uL. The reaction was stopped after 15 min [~90% conversion (data not shown)] by the addition of 4 uL of an FeXO solution (final concentrations of 200 uM XO. |
| Affinity data for this assay | |
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