| Assay Method Information | |
| | AlphaLISA Enzyme Inhibitory Assay |
| Description: | General Procedure for PRMT5/MEP50 AlphaLISA Enzyme Inhibitory Assays: Assays were performed in the buffer consisting of 50 mM Tris, pH 8.5, 5 mM MgCl2</sub>, 50 mM NaCl with 0.01% Tween20 and 1 mM DTT added right before the assay. 2.5 μL of compound solution in the assay buffer with 4% DMSO and 5 μL of PRMT5/MRP50 complex/SAM mixture solution in the assay buffer which was pre-incubated for 30 minutes were added into a white low volume 384 well microtiter plate. This mixture solution was incubated for 15 minutes with gentle shaking at room temperature. Methyl transfer reaction was initiated by adding 2.5 μL of Biotin-H4 (1-21) peptide substrate solution in the assay buffer. Final concentrations of PRMT5/MEP50, SAM, Biotin-H4 peptide substrate, and DMSO were 25 nM, 10 μM, 30 nM, and 1%, respectively. The reaction was allowed to perform for 120 minutes in dark with gentle shaking at room temperature after which 5 μL of Anti-H4R3-Me AlphaLISA acceptor beads in detection buffer from the manufacturer was added into the reaction mixture followed by incubation for 60 minutes. 10 μL of Streptavidin labeled AlphaLISA donor beads in detection buffer was added into the mixture followed by 30 minute incubation. Final acceptor and donor beads concentrations were 10 μg/mL. Plates were read on an Envision multimode plate reader from PerkinElmer (Waltham Mass., USA) with an excitation wavelength of 680 nm and emission wavelength of 615 nm. IC50 values of inhibitors were obtained by fitting the fluorescence intensity vs inhibitor concentrations in a sigmoidal dose-response curve (variable slopes, four parameters) using Prism 7. |
| Affinity data for this assay | |
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