| Assay Method Information | |
| | PRMT5 Biochemical Assay |
| Description: | The assay was carried out in 384-well low volume black plates in a reaction mixture containing 10 nM PRMT5/MEP50 complex, biotinylated histone H4 peptide, 3 μM S-adenosylmethionine and 0-10 μM compound in buffer containing 50 mM Tris-HCl buffer (pH 8.5), 0.005% BSA, 1 mM TCEP and 0.002% Tween-20. The PRMT5/MEP50 enzyme was incubated with compounds disclosed herein and biotinylated histone H4 peptide for 20 minutes at room temperature. The reaction was initiated by addition of S-adenosylmethionine. After reacting at room temperature for 120 minutes, the detection solution containing Eu-labeled antibody and dye-labeled acceptor in detection buffer was added to the reaction mixture. Plates were sealed and incubated at room temperature for 60 minutes, and the TR-FRET signals (excitation 337 nm, emission 665/620 nm) were recorded on a PHERAstar FSX plate reader (BMG Labtech). The inhibition percentage of PRMT5/MEP50 activity in presence of increasing concentrations of compounds was calculated based on the ratio of fluorescence at 665 nm to that at 620 nm. The concentration of MTA is 800nM. |
| Affinity data for this assay | |
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