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Assay Method Information |
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| | Inhibition Assay |
| Description: | Compound 75 was serially diluted 3 fold in DMSO for 10 points and 1 μL , was plated in a 384 well microtiter plate. Positive control (100% inhibition standard) was 100 μM final concentration of SAH and negative control (0% inhibition standard) contained 1 μL, of DMSO. Compound 75 was then incubated for 30 minutes with 40 μL per well of EZH2 wild-type and mutants at 8 nM in pH 7.6 assay buffer (20 mM BICINE, 100 mM KC1, 1 mM DTT, 0.002% TWEEN 20 (generic: polysorbate 20), 0.005% BSG). A substrate mix at 10 μL per well was added which contained S-adenosylmethionine-Cl (SAM) at 150 nM and tritiated SAM at 100 nM, and biotinylated oligonucleosome at 150 nM in pH 7.6 assay buffer. Quenched enzyme reaction was transferred to a streptavidin-coated FLASHPLATE (Perkin Elmer, catalog number SMP410), allowed to bind for one hour, and detected on a TOPCOUNT NXT HTS (Perkin Elmer). |
| Affinity data for this assay | |
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