| Assay Method Information | |
| | Monoamine Oxidase A (MAO-A) Selectivity Assay |
| Description: | MAO-Glo Assay Kit: Promega, Cat No. V1402MAO-A Enzyme: Promega, Cat No. V1452Clorgyline: Sigma, Cat No. M3778 (50 mg)Assay Plates:96 well white polystyrene, flat bottom plates, no lid: Fisher, Cat No. DPS-134-050AInhibitors (Test compounds):10 mM stock diluted to 10, 3, 1, 0.3, 0.1, 0.03, 0.01, 0.003, 0.001, and 0.0003 mM in drug plate, resulting in a concentration of 100, 30, 10, 3, 1, 0.3, 0.1, 0.03, 0.01, and 0.003 uM in the assay.Procedure:1. Dilute MAO substrate 1:50 with MAO A reaction buffer and add 25 μl to each well of a 96 well plateRequire 2.8 ml: 56 μl MAO substrate+2744 μl reaction buffer2. Add 0.5 μl test compound serial dilutions, in duplicate3. Add 0.5 μl serial dilutions of Clorgyline (positive control) down column 11 (no duplication)4. Add 25 μl specific MAO enzyme dilution:dilute 1:520 with MAO-A specific reaction buffer (5.3 μl MAO-A+2750.7 μl reaction buffer)5. Add following controls, in duplicate, down column 12:No Luciferin: 25 μl MAO enzymeDMSO: 0.5 μl DMSO+25 μl MAO enzymeBlow Out: 1 μl undiluted MAO-A enzyme+24 μl reaction buffer6. Incubate for 1 hour at room temp on a plate shaker7. Add 50 μl Luciferin detection reagent to each well EXCEPT no luciferin control wells (add 50 μl reaction buffer)8. Incubate for 20 min at room temp on a plate shaker, protected from light9. Measure the luminescence using a plate reader (read mode: luminescence 500 ms/well) |
| Affinity data for this assay | |
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