| Assay Method Information | |
| | Scintillation Proximity Assay |
| Description: | 1. Prepare 10 mM stock of compounds from solid in 100% DMSO.2. Set up an 11-point serial dilution (1:4 dilution, top concentration 10 mM) in 100% DMSO for each test compound in a 384 well plate leaving columns 6 and 18 for DMSO controls.3. Dispense 10 nL of compound from the dilution plate into reaction plates (Corning, 384-well polystyrene NBS, Cat #3673).Part B. Reagent PreparationPrepare the following solutions:1. 1× Base Buffer, 50 mM Tris-HCl, pH 8, 2 mM MgCl2: Per 1 L of base buffer, combine 1 M Tris-HCl, pH 8 (50 mL), 1 M MgCl2 (2 mL), and distilled water (948 mL).2. 1× Assay Buffer: Per 10 mL of 1× Assay Buffer, combine 1× Base Buffer (9.96 mL), 1 M DTT (40 uL), and 10% Tween-20 (1 uL) to provide a final concentration of 50 mM Tris-HCl, pH 8, 2 mM MgCl2, 4 mM DTT, 0.001% Tween-20.3. 2× Enzyme Solution: Per 10 mL of 2× Enzyme Solution, combine 1× Assay Buffer (9.99 mL) and 3.24 uM EZH2 5 member complex (6.17 uL) to provide a final enzyme concentration of 1 nM.4. SPA Bead Solution: Per 1 mL of SPA Bead Solution, combine Streptavidin coated SPA beads (PerkinElmer, Cat # RPNQ0261, 40 mg) and 1× Assay Buffer (1 mL) to provide a working concentration of 40 mg/mL.5. 2× Substrate Solution: Per 10 mL of 2× Substrate Solution, combine 40 mg/mL SPA Bead Solution (375 uL), 1 mM biotinylated histone H3K27 peptide (200 uL), 12.5 uM 3H-SAM (240 uL; 1 mCi/mL), 1 mM cold SAM (57 uL), and 1× Assay Buffer (9.13 mL) to provide a final concentration of 0.75 mg/mL SPA Bead Solution, 10 uM biotinylated histone H3K27 peptide, 0.15 uM 3H-SAM ( 12 uCi/mL 3H-SAM), and 2.85 uM cold SAM.6. 2.67× Quench Solution: Per 10 mL of 2.67× Quench Solution, combine 1× Assay Buffer (9.73 mL) and 10 mM cold SAM (267 uL) to provide a final concentration of 100 uM cold SAM.Part C. Assay Reaction in 384-well Grenier Bio-One PlatesCompound Addition1. Stamp 10 nL/well of 1000× Compound to test wells (as noted above).2. Stamp 10 nL/well of 100% DMSO to columns 6 & 18 (high and low controls, respectively).Assay1. Dispense 5 uL/well of 1× Assay Buffer to column 18 (low control reactions).2. Dispense 5 uL/well of 2× Substrate Solution to columns 1-24 (note: substrate solution should be mixed to ensure homogeneous bead suspension before dispensing into matrix reservoir).3. Dispense 5 uL/well of 2× Enzyme Solution to columns 1-17, 19-24.4. Incubate the reaction for 60 min at room temperature.Quench1. Dispense 6 uL/well of the 2.67× Quench Solution to columns 1-24.2. Seal assay plates and spin for 1 min at 500 rpm.3. Dark adapt plates in the ViewLux instrument for 15-60 min. |
| Affinity data for this assay | |
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