| Assay Method Information | |
| | MTase-Glo Methyl Transferase Assay |
| Description: | Table 1: The PRMT5 inhibitory activity of test compounds was determined using the MTase-Glo™ assay (Promega), which monitors the product (S-adenosyl homocysteine or SAH) of methyltransferase reactions. The PRMT5 MTase-Glo assays were conducted in a 384-well white ProxiPlate (PerkinElmer, catalog no.: 6008280) in a total volume of 12 μL. The PRMT5 enzymatic reaction (in 4 μL) contained 50 nM PRMT5/MEP50 (Reaction Biology Corp, catalog no.: HMT-22-148), 25 μM S-adenosyl methionine (SAM, Promega), 5 μM Histone H4 peptide (1-21) (BPS Bioscience, catalog no.: 52018-2) and five-fold serially diluted compounds in a reaction buffer of 50 mM Tris (pH 8.0), 50 mM NaCl, 0.01% Tween 20, 0.01% BSA, and 1 mM DTT. The test compounds were pre-incubated with PRMT5/MEP50 and Histone H4 peptide for 20 minutes at room temperature before the addition of SAM to initiate the PRMT5 reaction. The reaction was allowed to proceed for 1 hour at 37° C. and was terminated by 2 μL of 3× MTase-Glo™ Reagent (Promega) and 150 μM EPZ015666 (Selleck, catalog no.: 1616391-65-1). After a 30-minute incubation at room temperature, 6 μL of MTase-Glo™ Detection Solution (Promega) was added and the plate was incubated at room temperature for an additional 30 minutes. The light signal corresponding to the amount of SAH produced by the PRMT5 reaction was subsequently measured using an Envision multimode reader (PerkinElmer). |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |