| Assay Method Information | |
| | MAT2A Enzymatic Testing Method |
| Description: | 1. Experimental Stepsa) 5×MAT2A assay-buffer (250 mM Tris-HCl, pH 8.0; 250 mM KCl; 75 mM MgCl2; 0.025% BSA; 0.05% Brij35; 1.5 mM EDTA) were first prepared and partially diluted to 1× for further use;b) Preparation and addition of MAT2A enzyme (BPS, 71401): MAT2A enzyme was prepared to 3.674 ng/μL (1.67×, final concentration 2.20 ng/μL) with 1×MAT2A assay-buffer. With BioTek (MultiFlo FX) automatic dispenser, 15 μL 1.67×MAT2A enzyme solution were added to the compound test well and negative control well respectively. At the same time, 15 μL of 1×MAT2A assay-buffer was added to the blank control well;c) Preparation and addition of compounds: the compound to be tested was diluted from 10 mM stock solution to 100 μM with DMSO. The positive drug AGI-24512 was diluted under the same conditions. Tecan compound titrator (D300e) was applied to automatically spray the solution into each well according to the preset concentration gradient, with the dispensed volume being extremely small and negligible. The starting concentration gradient is 1 μM, ½ log, and a total of 8 gradients are set. The plate was centrifuged at 2500 rpm for 30 s and incubated at 25° C. for 30 min;d) Preparation of ATP: 10 mM ATP (Sigma, A7699) was diluted to 700 μM with 1×MAT2A assay-buffer for further use;e) Preparation and addition of substrate and ATP mixture: 5×MAT2A assay-buffer, 3 μL/well; 750 μM L-methionine (Adamas, 01100469), 2.5 μL/well; 700 μM ATP, 2.5 μL/well; double distilled water, 2 μL/well. With total amount of mixed solution required determined according to the number of detection wells, using BioTek (MultiFlo FX) automatic dispenser, 10 μL of the mixed solution was added to each well; The plate was centrifuged at 2500 rpm for 30 s and reacted at 25° C. for 150 min;f) Addition of Biomol Green detection reagent: with BioTek (MultiFlo FX) automatic dispenser, 50 μL Biomol Green (Enzo, BML-AK111) was added to each well, the plate was centrifuged at 2500 rpm for 30 s and incubated at 25° C. for 20 min;g) Upon completion of the reaction, the OD620 value was obtained using Perkin Elmer (Envision 2105) multifunctional plate reader. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |