| Assay Method Information | |
| | PRMT5 Inhibitory Activity Assay In Vitro |
| Description: | 1. Enzyme Reaction Process(1) 1× Assay buffer (modified Tris buffer) was configured.(2) Dilute compound: the compound was dissolved in 100% DMSO and the compound solution was added to a 384-well plate using Echo 550.(3) Configure enzyme solution: PRMT5 was added to 1× assay buffer to prepare enzyme solution 1; PRMT5 and MTA was added to 1× assay buffer to prepare enzyme solution 2.(4) Configure substrate solution: peptide segments and [3H]—SAM was added to 1× assay buffer.(5) 15 μL of enzyme solution was added to the 384-well plate, and 15 μL of 1× assay buffer was added to the negative control well, and incubated at room temperature for 30 minutes.(6) 15 μL of substrate solution was added to each well, and incubated at room temperature for 90 minutes.(7) Configure termination reaction solution: pre-cooled SAM was added to 1× assay buffer.(8) 10 μL of termination reaction solution was added to each well to terminate the reaction.(9) 25 μL/well of the mixed solution was transferred to Flashplate and incubated for 1 hour at room temperature.(10) the Flashplate three was washed times with dH2O+0.1% Tween-20 solution.(11) the radiometric values was readed with Microbeta. |
| Affinity data for this assay | |
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