| Assay Method Information | |
| | Inhibition Assay |
| Description: | KDM1A inhibition assay: a multistep enzymatic reaction in which the enzyme first produces H2O2 during the demethylation of lysine 4 in a 21 AA H3K4me2 N-terminal peptide. KDM1A chemoprobes were pre-incubated for 15 min with human recombinant KDM1A enzyme (BPS Bioscience, Ref. 50100) on ice in the assay buffer (50 mM sodium phosphate pH 7.4). The enzymatic reaction was initiated by the addition of KM dimethylH3K4 peptide substrate (Anaspec, Ref. 63677). After 30 min of incubation at 37° C. Amplex Red reagent and the horseradish peroxidase (HRP) solution were added according to the recommendations of the supplier (Invitrogen) and left to incubate for 5 min at room temperature in the dark. Conversion of the Amplex Red reagent to resorufin, was monitored by fluorescence (λex=540 nm, λem=590 nm) using a microplate reader (Infinite F200 Tecan). Signals were corrected for background and the IC50 value was calculated with GraphPad Prism Software. |
| Affinity data for this assay | |
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