| Assay Method Information | |
| | AOC2 Biochemical Assay |
| Description: | An AOC2 enzyme containing cell homogenate is generated by transient transfection of 6×106 HEK293 cells per flask (T75) with 9 μg pCMV-SPORT6-AOC2 (BC142641rc, #pCS6(BC142641)-seq-TCHS1003-GVO-TRI, BioCat) in 750 μL of EMEM culture medium (#BE12-611F, Lonza) and 33,75 μl Attractene (#301005, Qiagen). Cells are cultured for 3 days in EMEM culture medium containing 10% FCS (#04-00-1A, Biological Industries). After washing twice with ice cold PBS, cells are lysed by mechanic homogenation and cleared supernatants are shock frozen in liquid nitrogen and stored at −80° C.For the determination of AOC2 enzymatic activity cell lysates are thawed on ice and 1:1 diluted with reaction buffer. An Aliquot of 45 μL is added to the compound dilution and incubated for 30 min at 37° C. The enzymatic reaction is started with the addition of 50 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), 2 mM phenylethylamine (#P6513-25G Sigma-Aldrich), 0.05% Pluronic F-127 (#P3000MP Sigma-Aldrich), pH 7.4, 37° C.). |
| Affinity data for this assay | |
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