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Biochemical enzyme assays
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Compound was incubated for 30 mins with 40 μL per well of 5 nM PRC2 (final assay concentration in 50 μL was 4 nM) in 1× assay buffer (20 mM Bicine [pH 7.6], 0.002% Tween-20, 0.005% Bovine Skin Gelatin and 0.5 mM DTT). 10 μL per well of substrate mix comprising assay buffer 3H-SAM, unlabeled SAM, and peptide representing histone H3 residues 21-44 containing C-terminal biotin (appended to a C-terminal amide-capped lysine) were added to initiate the reaction (both substrates were present in the final reaction mixture at their respective Km values, an assay format referred to as “balanced conditions”). The final concentrations of substrates and methylation state of the substrate peptide were indicated for each enzyme. Reactions were incubated for 90 mins at room temperature and quenched with 10 μL per well of 600 μM unlabeled SAM. The mixtures were then transferred to a 384-well flashplate and washed after 30 mins.
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References:
[1]. Knutson SK, Wigle TJ, Warholic NM, Sneeringer CJ, Allain CJ, Klaus CR, Sacks JD, Raimondi A, Majer CR, Song J, Scott MP, Jin L, Smith JJ, Olhava EJ, Chesworth R, Moyer MP, Richon VM, Copeland RA, Keilhack H, Pollock RM, Kuntz KW. A selective inhibitor of EZH2 blocks H3K27 methylation and kills mutant lymphoma cells. Nat Chem Biol. 2012 Nov;8(11):890-6.
[2]. Chen L, Wu Y, Wu Y, Wang Y, Sun L, Li F. The inhibition of EZH2 ameliorates osteoarthritis development through the Wnt/β-catenin pathway. Sci Rep. 2016 Aug 19;6:29176.
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