| Assay Method Information | |
| | Scintillation Proximity Assay (SPA) |
| Description: | Compounds contained herein were evaluated for their ability to inhibit the methyltransferase activity of EZH2 within the PRC2 complex. Human PRC2 complex was prepared by co-expressing each of the 5 member proteins (FLAG-EZH2, EED, SUZ12, RbAp48, AEBP2) in Sf9 cells followed by co-purification. Enzyme activity was measured in a scintillation proximity assay (SPA) where a tritiated methyl group is transferred from 3H-SAM to a lysine residue on a biotinylated, unmethylated peptide substrate derived from histone H3. The peptides were captured on streptavidin-coated SPA beads and the resulting signal was read on a ViewLux plate reader. 4. Prepare 10 mM stock of compounds from solid in 100% DMSO. 5. 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. 6. Dispense 10 nL of compound from the dilution plate into reaction plates (Corning, 384-well polystyrene NBS, Cat#3673). Part B. Reagent Preparation Prepare the following solutions: 7. 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). 8. 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. 9. 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. 10. 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. 11. 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. 12. 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 Plates Compound Addition 3. Stamp 10 nL/well of 1000× Compound to test wells (as noted above). 4. Stamp 10 nL/well of 100% DMSO to columns 6 & 18 (high and low controls, respectively). Assay 10. Dispense 5 uL/well of 1× Assay Buffer to column 18 (low control reactions). 11. 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). 12. Dispense 5 uL/well of 2× Enzyme Solution to columns 1-17, 19-24. 13. Incubate the reaction for 60 min at room temperature. Quench 5. Dispense 6 uL/well of the 2.67× Quench Solution to columns 1-24. 6. Seal assay plates and spin for 1 min at 500 rpm. 7. Dark adapt plates in the ViewLux instrument for 15-60 min. Read plates 2. Read the assay plates on the Viewlux Plate Reader utilizing the 613 nm emission filter or clear filter (300 s exposure). Reagent addition can be done manually or with automated liquid handler. Results |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |