| Assay Method Information | |
| | Enzymatic Activity Test |
| Description: | The specific steps were as follows: The compounds having the test starling concentration of 1 uM or 10 uM were diluted into 10 concentration points in a 3-fold gradient. 250 nL of the solutions of the compounds to be tested with 10 different concentrations were taken and added into a 384 well plate for later use. 20 ug/mL of MAT2a enzyme solution was prepared with Assay buffer (50 mM Tris, 50 mM KCl, 10 mM MgCl2, 0.05% polyoxyethylene lauryl ether, pH 8.0). 15 uL of the MAT2a enzyme solution at 20 ug/mL was add into the wells of the compounds to be tested at different concentrations; and 15 uL of Assay buffer was added into the negative control well. An incubation was carried out for 15 minutes after shaking for mixing well. A mixed substrate solution (comprising 400 uM ATP and 600 uM L-Methionine) was prepared with Assay buffer. 10 uL of the mixed substrate solution was added to the positive control well, the compound to be tested well, and the negative control well, respectively, and the reaction began, for a reaction time of 150 min. Then, 50 uL of the reaction stop solution (BIOMOL Green Reagent Enzo lifesciences, Cargo No. BML-AK111-1000) was added to stop the reaction, followed by being centrifuged at 1000 rpm for 60 s and then being incubated for 15 min. OD620 was read and data were processed. |
| Affinity data for this assay | |
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