| Assay Method Information | |
| | In vitro Evaluation of Kinase Activity Assay |
| Description: | Experimental Method2.1. Preparation of reaction buffer and reaction stop solution: the composition of 1× reaction buffer was 10 mM Tris-HCl, pH 8.0; 0.01% Tween-20; and 1 mM DTT. The composition of the reaction stop solution was 125 μM 3H-SAM solution.2.2 Formulation of Compounds2.2.1 Dilution of CompoundsThe compounds were dissolved in 100% DMSO to prepare 10 mM stock solutions and then diluted to desired concentrations on an Echo 384 well plate.2.2.2 Transfer of Compounds to 384 Well Reaction Plate250 nL of the compounds diluted above were transferred from the Echo 384 well plate to a 384 well reaction plate using the Echo550 instrument.2.3 Enzymatic Reaction2.3.1 Preparation of 1.67× Enzyme SolutionPRMT5 was added to 1× reaction buffer to form 1.67× enzyme solution.2.3.2 Preparation of 2.5× Substrate SolutionThe polypeptide substrate and [3H]-SAM were added to 1× reaction buffer to form 2.5× substrate solution (the final concentrations were 100 nM and 250 nM, respectively).2.3.3 Addition of Enzyme Solution to 384 Well Plate15 μL of 1.67× enzyme solution was added to the wells of a 384 well reaction plate. For control wells without enzymatic activity, the enzyme solution was replaced with 15 μL of 1× reaction buffer. The plate was centrifuged at 1000 rpm for 1 min and incubated at room temperature for 15 min.2.3.4 Addition of Substrate Solution to 384 Well Plate to Initiate Enzymatic Reaction 10 μL of 2.5× substrate solution was added to each well of the 384 well reaction plate. The plate was centrifuged at 1000 rpm for 1 min. The reaction was carried out at 25° C. for 60 min. |
| Affinity data for this assay | |
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