| Assay Method Information | |
| | Biochemical Assay |
| Description: | The brief introduction of the test principle: L-methionine and ATP can be converted into SAM, inorganic phosphate, and inorganic diphosphate under the catalysis of MAT2A enzymes. The content of inorganic phosphate in the sample can be quantitatively determined by adding a developer such as ammonium molybdate or the like to the enzymatic reaction mixture, thereby reflecting the enzymatic activity of MAT2A. Material: MAT2A screening kits were purchased from BPS bioscience (U.S.); 384-well plates were purchased from Corning Company (U.S.).1. MAT2A protein (Pharmaron Beijing Co., Ltd.);2. L-methionine (Sigma #M9625-5G)3. ATP (Sigma #A7699-1G)4. KCl (Sigma #60142-500ML-F)5. Tris (Sigma #T2663-1L)6. MgCl2 (Sigma #M1028)7. EDTA (Invitrogen #AM9260G)8. BSA (Sangon Biotech #A500023-0100)9. PiColorLock (abcam #ab270004)Detection method: In the experimental group, the test compound was dissolved in DMSO, diluted to a final concentration of 10 μM using Echo, and diluted 3-fold. 80 nL of the dilution was transferred to a 384-well plate.The experimental buffer (50 mM Tris, 50 mM KCl, 15 mM MgCl2, 100 μM EDTA, 0.005% BSA) was prepared. The MAT2A protein was diluted with the experimental buffer (at final concentration of 4 Vg/mL). 40 μL of 2 MAT2A solution was added to the 384-well plate. The plate was centrifuged at 1000 rpm for 1 min and incubated at room temperature for 120 min.L-methionine and ATP (the final concentration of L-methionine was 200 JIM and the final concentration of ATP was 400 μM) were diluted with the experimental buffer. 40 μL of 2 L-methionine and ATP solution was added to initiate the reaction. The plate was centrifuged at 1000 rpm for 1 min and incubated at room temperature for 90 min.The PiColorLock reaction catalyst was mixed with the PiColorLock buffer at a ratio of 1:100 according to the instructions. 20 μL was added to each well, and the plate was shaken for 30 s. 8 μL stabilizing reagent was added, and the plate was shaken for 30 s. The signal values were measured after 30 min of incubation at room temperature. |
| Affinity data for this assay | |
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