| Assay Method Information | |
| | AlphaLISA Assay |
| Description: | Reactions were performed in 50 mM HEPES [pH 7.2 for KDM5 or pH 7.5 for KDM4A(1-350)] containing 0.01% BSA and 0.01% Tween-20. To each well, 3 μL of enzyme mix (final 10 nM), containing either full-length KDM5B, KDM5A, KDM5C, KDM5B(1-755)ΔAP or KDM4A(1-350), was added using a BioRAPTR flying reagent dispenser (FRD; Beckman Coulter, Fullerton, CA) while buffer alone served as a negative control. A Kalypsys pin-tool was then employed to transfer 23 nL of compound solutions in DMSO to each well, yielding a final concentration range up to 57 μM. Following a 10 min incubation at room temperature, 1 μL of 4X substrate mix containing b-H3K4me3 peptide substrate (final 100 nM), αKG (final 25 μM), (NH4)2Fe(SO4)2 (final 50 μM) and ascorbic acid (final 10 μM) was added to initiate the reaction. Reactions were allowed to proceed at room temperature for 45 min. To detect demethylated b-H3K4me3 peptide product, reactions were incubated with 20 μg/mL antim |
| Affinity data for this assay | |
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