| Assay Method Information | |
| | KAT Assay Protocol |
| Description: | 1. Final reaction conditions for each KAT assay in a 20 ul assay reaction volume:i. KAT6A 15 nM, 1 uM AcCoA, 2 uM H3 1-21 peptide, 45-minute reaction2. Add 0.5 ul of diluted compound to the assay plate (384-well V-bottom polypropylene plates) or 0.5 ul of DMSO for control wells.3. Add 10 ul of 2× Histone peptide/2× AcCoA mix to the assay plate.4. Add 10 ul of 2× enzyme to the assay plate.5. Stop the reaction after the indicated time with the addition of 2 ul of 5% formic acid6. Each reaction was analyzed using self-assembled monolayer desorption/ionization time-of-flight mass spectrometry (Mrksich, Milan (2008) Mass Spectrometry of Self-Assembled Monolayers: A New Tool for Molecular Surface Science ACS Nano 2008 2 (1), 7-18; SAMDI Tech, Inc. (Chicago, IL)).7. Area under the curve (AUC) for both substrate and product peaks was determined for KAT6A at M.W. 2723 [Substrate+H]+ and 2765 [Product+H]+ with a +/−1 Da tolerance, respectively.8. Percent conversion to product was calculated by: AUCProduct/(AUCSubstrate+AUCProduct). |
| Affinity data for this assay | |
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