| Assay Method Information | |
| | AOC1 Biochemical Assay |
| Description: | For the determination of AOC1 activity or compound AOC1 inhibition potency, the compound inhibitors are dissolved in DMSO and adjusted to the respective assay concentration with reaction buffer (100 mM sodiumphosphate, 0.05% Pluronic F-127 (#P3000MP Sigma-Aldrich), pH 7.4). An aliquot of 3 μL of the compound dilution is added to a 384 well plate (Optiplate, PS, flat bottom F, black, PERKIN ELMER, #6007270) in a DMSO concentration of 6.6%.An AOC1 enzyme aliquot (#8297-AO-010, R&D Systems) is thawed on ice, diluted in reaction buffer and added in a volume of 7 μL to the wells to give a final assay concentration of 1 ng/well. After incubation of inhibitor and enzyme for 30 minutes at 37° C., the enzymatic reaction is started with the addition of 10 μL of Amplex® Red reaction mix (final assay concentration: 100 mM sodiumphosphate, 120 μM Amplex® Red reagent (#A22177 Molecular Probes), 1.5 U/mL Horseradish Peroxidase (#P8375 Sigma-Aldrich), 200 μM putrescine (#P7505 Sigma-Alrdich), 0.05% Pluronic F-127 (#P3000MP Sigma-Aldrich), pH 7.4, 37° C.).After an incubation for 30 minutes at 37° C. the turnover of the substrate is determined directly (or after the addition of an excess of an amine-oxidase inhibitor) with a fluorescence reader (Ex 540 nm/Em 590 nm) like Envision 2104 Multilabel Reader (PERKIN ELMER). |
| Affinity data for this assay | |
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