| Assay Method Information | |
| | Scintillation Proximity Assay (SPA) |
| Description: | Protocol 2: 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.Part A. Compound Preparation 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). Percent inhibition was calculated relative to the DMSO control for each compound concentration and the resulting values were fit using standard IC50 fitting parameters within the ABASE data fitting software package. |
| Affinity data for this assay | |
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