| Assay Method Information | |
| | The radiometric assay for PRMT5 based on FlashPlate technology was adopted to determine IC50 of the test compound. |
| Description: | After dissolved in dimethyl sulfoxide respectively, the test compounds were added into an Echo Qualified 384-well plate and diluted to the desired concentrations. The diluted test compounds were transferred from the Echo Qualified 384-well plate to a 384-well reaction plate using an Echo 550 instrument, and dimethyl sulfoxide was transferred into both the control and blank wells. PRMT5 was added to 1×reaction buffer (including 10 mM Tris-HCl; pH 8.0; 0.01% Tween-20; 1 mM DTT) to form a 1.67×enzyme solution (at an enzyme concentration of 5 nM). A polypeptide substrate and [3H]-SAM were added to 1× reaction buffer to form a 2.5× substrate solution (the terminal concentrations of the substrates were 100 nM and 250 nM, respectively). At a volume of 15 μL/well, the 1.67× enzyme solution was added into wells of the 384-well reaction plate. In case of the blank wells, the enzyme solution was replaced with 15 μL of the 1× reaction buffer. The reaction plate was centrifuged at 1000 rpm for 1 min, and incubated at room temperature for 15 min. To each well of the 384-well reaction plate, 10 μL of the 2.5× substrate solution was added, centrifuged at 1000 rpm for 1 min, and reacted at 25° C. for 60 min. To each well of the 384-well reaction plate, 5 μL of reaction stop solution (which was 125 M cold SAM solution) was added to terminate the reaction. From each well of the test plate, 25 μL was measured and transferred to Flashplate and left at room temperature for 1 h. Thereafter, the Flashplate was washed with 0.1% Tween-20 solution three times. Readings were taken with MicroBeta 2. |
| Affinity data for this assay | |
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