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| | PRC2 Biochemical Assay |
| Description: | To assess the compounds potency in the H3K27me0 peptide (21–44)-based PRC2 enzymatic assay, compounds were serially diluted three-fold in DMSO to obtain a total of twelve concentrations. Then, compounds at each concentration were transferred by Mosquito into a 384-well PerkinElmer ProxiPlate 384 plus plate. The typical reaction (12 μl) includes 1 μM SAM (at Km), 1.5 μM H3K27me0 peptide (at Km) and 10 nM PRC2 in the assay buffer composed of 20 mM Tris–HCl, pH 8.0, 0.01% Triton X-100, 0.5 mM DTT and 0.1% BSA. The reactions were stopped by adding 3 μl quench solution containing 2.5% TFA and 320 nM SAH-d4 (Cayman chemical). The procedure for the PRC2 LC–MS assay with recombinant nucleosome core particles (NCP) as substrate was very similar to the H3K27me0 peptide based LC–MS assay described above except NCP was used as substrate. Meanwhile, activation peptide H3K27me3 (21–44) was included to stimulate the PRC2 activity against nucleosome substr |
| Affinity data for this assay | |
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