| Assay Method Information | |
| | Test Method for PRMT5 MTA Enzyme Activity |
| Description: | 1× test buffer (10 mM Tris, 1 mM DTT, 0.01% BSA, 0.01% Tween-20, adjusted to pH 8.0) was prepared. The test compound was dissolved in DMSO to prepare a 10 mM mother liquor, and the mother liquor was diluted to a 100-fold final concentration. 100 nL of the compound was added to each well of the administration group, and 100 nL of DMSO solution was added to blank wells and negative control wells. Then, 5 μL of a solution of PRMT5/MEP50 (BPS, Cat #31921) (final concentration: 1 nM) and MTA (final concentration: 1 μM) was added to the wells of the administration group and negative control wells, and 5 μL of 1× test buffer was added to the blank wells. Incubation was performed at room temperature for 15 minutes. 5 μL of a mixed solution of H4(1-21)S1ac (0.035 μM; GL Biochem, customized) and SAM (0.434 μM; Sigma, Cat #A7007) was added to each well and incubated at room temperature for 1 hour. 1× Epigenetics buffer (PerkinElmer, Cat #AL008F) was prepared and used for diluting acceptor microbeads and donor microbeads. 15 μL of acceptor microbeads (final concentration: 10 μg/mL; PerkinElmer, Cat #AL150C) and donor microbeads (final concentration: 10 μg/mL; PerkinElmer, Cat #AS106M) were added and incubated at room temperature for 1 hour in the dark. Then, detection was performed using the Alpha module of the reader Enspire microplate. |
| Affinity data for this assay | |
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