| Assay Method Information | |
| | KDM5 TR-FRET Assay |
| Description: | Time-resolved fluorescence resonance energy transfer (TR-FRET) assays were carried out using full-length KDM5 enzymes in 384-well black ProxiPlates (PerkinElmer Inc.). The indicated final reaction concentration of KDM5A (2 nM), KDM5B (8 nM) or KDM5C (4 nM) was combined with 2-OG (Sigma) at Km (final reaction concentration of 1 μM for KDM5A and KDM5C and of 2 μM for KDM5B, unless otherwise indicated (20× = 20 μM)) in assay buffer (50 mM HEPES, pH 7.0, 2 mM DTT, 0.5 mM ascorbic acid and 0.01% (vol/vol) Triton-X-100) in a total volume of 2.5 μl. To this we added 2.5 μl of compound that had previously been diluted in ten-point serial dilutions in DMSO and then diluted in assay buffer to 1% DMSO (vol/vol, final). Reaction was initiated by the addition of 5 μl of 200 nM H3K4me3 peptide substrate stock (ARTK(me3)QTARKSTGGKAPRKQLA-NovaTagPEG-biotin, New England Peptide) in assay buffer containing 200 μM (NH4)2Fe(SO4)2 hexahydrate (Sigma). Reactions were carr |
| Affinity data for this assay | |
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