| Assay Method Information | |
| | Biological Assay (10X SAM) |
| Description: | A. Compound preparation1. Prepare 10 mM stock solutions in 100% DMSO from solid material2. Serial dilute 10 mM compound stocks either 2 or 3-fold in 100% DMSO to generate compounds for 11 point dose responseB. Reagent preparation1. Prepare 1x assay buffer containing 100 mM Tris pH 8.5, 4 mM DTT and 0.01% Tween-202. Dilute purified HeLa oligonucleosomes and recombinant histone H1 (New England Biolabs) in assay buffer to 1.67x.3. Dilute PRC2 4 protein complex (EZH2, EED, SUZ12, RbAp48) to 3.5x in assay buffer4. Prepare 10x3H SAM solution in assay buffer using 0.94 uCi/well of radioactive SAM (Perkin Elmer) and sufficient non-labeled SAM (Sigma) for 15 uM final concentration.5. Dilute TCA to 20% in DI waterC. Enzyme reaction1. Final reaction conditions are PRC2 4-protein complex at 4 nM when using WT EZH2 or 6 nM when using Y641N mutant EZH2, 1.5 uM SAM, 25 ug/mL oligonucleosomes, 50 nM rH1 in a 50 ul reaction volume.2. Add 1 ul of diluted compound to the assay plate (96-well V-bottom polypropylene plates) or 1 ul of DMSO for control wells.3. Add 30 ul of nucleosomes to the assay plate4. Add 14 ul of either WT or Y641N mutant PRC2 4 protein complex to the assay plate5. Add 5 ul of 3H SAM to start the reaction.6. Stop the reaction after 60 minutes with the addition of 100 ul of 20% TCA7. Transfer 150 ul of quenched reaction into a prepared filterplate (Millipore # MSIPN4B10)8. Apply vacuum to the filterplate to filter the reaction mix through the membrane.9. Wash the filterplate with 5x200 ul of PBS, blot dry and dry in an oven for 30 minutes10. Add 50 ul of microscint-20 scintillation fluid (Perkin Elmer) to each well, wait 30 minutes and count on a liquid scintillation counter. |
| Affinity data for this assay | |
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