| Assay Method Information | |
| | PRMT5/MEP50 HotSpot Methyltransferase Assay |
| Description: | Table 2: The assay used recombinant full-length histone H2A as the substrate of PRMT5. Enzymatic transfer of the tritiated methyl group from S-adenosyl-L-[methyl-3H]methionine (3H-SAM) to the histone H2A protein generated a radiolabeled histone H2A by measuring in a scintillation counter to determine the activity of PRMT5 enzyme in the presence and absence of compound. The assay reactions were conducted in the presence of 100 nM MTA. Briefly, compounds were solubilized in 100% DMSO at the highest concentration of 10 mM. For IC50 determinations, the initial starting concentration for the serial dilutions of each compound is 50 μM. Control samples lacking compound, PRTM5/MEP50 complex or various reaction components also were prepared and processed in parallel with compound test samples. SAH was used as a positive control for assay validation. To measure PRMT5 inhibitory activity, 1 nM PRTM5/MEP50 complex was preincubated with test compound in assay buffer containing 5 μM full-length histone H2A for 15 minutes at room temperature. The enzymatic reaction was initiated by adding 1 μM 3H-SAM (final concentration) and the mixture was incubated at 30° C. for 1 hour. The reaction was stopped and transferred to filter paper for detection. The amount of tritiated H2A in each sample was determined using a scintillation counter. |
| Affinity data for this assay | |
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