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| | KDM4C Enzymatic Screening Assay |
| Description: | High-throughput MS assays were carried out using truncated KDM4C in 384-well V-bottom polypropylene plates (Greiner, Inc.). His-tagged recombinant KDM4C (1-350) was overexpressed and purified in-house from Escherichia coli BL21(DE3) to near-homogeneity. The demethylation reaction buffer contained 50 mM Tris-HCl, pH 7.5, 0.01% Triton X-100, 5% glycerol, 1 mM sodium ascorbate (Sigma), 5 μM 2-OG (Sigma) and 20 μM Fe2(NH4)2(SO4)2 (Sigma). Compounds were added to the plates from 10 mM DMSO stocks either as mixtures of eight compounds for screening (25 nl each, 10 μM final) or as serial dilutions of single compounds for IC50 determinations. Additional DMSO was added as a backfill (final DMSO: 0.8% (vol/vol)) using a LabCyte Echo 550. In 25-μl demethylation reactions, 400 nM recombinant KDM4C and 20 μM H3K9me3 peptide (1-21 amino acids, New England Peptide) were incubated with compounds for 10 min, and then 2-OG and Fe2(NH4)2(SO4)2 were added to initiate the reaction |
| Affinity data for this assay | |
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