| Assay Method Information | |
| | Enzymatic Activity of MAT2A |
| Description: | 1. 1×Assaybuffer (buffer composition: Tris, KCl, MgCl2, BSA, Brij35, and EDTA; the solvent was ultrapure water) was prepared. MAT2A his-tag enzyme solution (containing 1.3 μL of MAT2A enzyme and 998.7 μL of 1×Assaybuffer per 1000 μL) and substrate mixed solution (containing 5 μL of ATP, 1.3 μL of L-methionine, and 993.7 μL of 1×Assaybuffer per 1000 μL) were prepared using 1×AssayBuffer.2. Preparation of compound concentration gradient: The test compound was tested with a starting concentration of 10 μM, and 10 equimolar concentrations were set according to a 3-fold dilution. Specifically, firstly, the compound was diluted into 10 different concentrations of series solutions in a 384 well plate (with final detection concentrations of 10, 3.33, 1.11, 0.37, 0.123, 0.041, 0.0137, 0.0046, 0.0015, and 0.0005 μM), and then 250 nL of the above series solutions was transferred to the 384 well reaction plate using an ultrasonic pipetting system Echo550 for later use. 250 nL of 100% DMSO was added into a negative control well and a positive control well, respectively. Double well test.3. 15 μL of MAT2A his-tag enzyme solution was added into both the compound well and the positive control well; and 15 μL of 1×Assaybuffer was added into the negative control well.4. The 384 well plate reaction plate was centrifuged at 1000 rpm for 60 seconds, shaken, mixed well, and incubated for 15 minutes. 10 μL of substrate mixed solution was added into each experimental well of the 384 well reaction plate to initiate the reaction.5. The 384 well reaction plate was centrifuged at 1000 rpm for 60 seconds, shaken, mixed well, and incubated for 150 minutes.6. 50 μL of enzyme reaction termination solution Biomol was added into all experimental wells of the 384 well reaction plate to terminate the reaction, centrifuged at 1000 rpm for 60 seconds, and incubated for 15 minutes. OD620 was Data analysis: the inhibition rate (%) of the compound was calculated and fitted to obtain the IC50 of the tested compound. |
| Affinity data for this assay | |
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