| Assay Method Information | |
| | Biological Assay |
| Description: | KAT2A/B Assay Protocol:A) Compound preparation1) Prepare 10 mM stock solutions in 100% DMSO from solid material.2) Serial dilute 10 mM, 1 mM, 0.1 or 0.01 mM compound stocks 3-fold in 100% DMSO for 11-point dose response.B) Reagent preparation1) Prepare 1×assay buffer containing 40 mM bis tris propane pH 8.0, 10 mM NaCl, 0.5 mM EDTA, and 0.002% Tween-20.2) Dilute Histone peptide (CPC Scientific) and KAT enzyme together in assay buffer to 1.25×.3) Dilute AcCoA (Sigma) in assay buffer to 5×.C) Enzyme reaction1) Final reaction conditions for each KAT assay in a 100ul assay reaction volume:i) KAT2A 0.5 nM, 1 uM AcCoA, 5 uM H3 1-21 peptide, 90-minute reactionii) KAT2B 0.5 nM, 1 uM AcCoA, 5 uM H3 1-21 peptide, 90-minute reaction2) Add 2 ul of diluted compound series to the assay plate (96-well round-bottom polypropylene plates) or 2 ul of DMSO for control wells.3) Add 11 μL of 10% formic acid to 100% effect (HPE) control wells.4) Add 80 ul of 1.25× Histone peptide/KAT mix to the assay plate via Multidrop Combi (ThermoFisher). Incubate 15 min at room temperature.5) Add 20 ul of 5× AcCoA to the assay plate via Multidrop Combi.6) Stop the reaction after the indicated time with the addition of 11 ul of 10% formic acid via Multidrop Combi.7) Each reaction was analyzed using Rapid Fire mass spectrometry platform. This methodology leverages an Agilent 6495 triple quadrupole (QQQ) mass spectrometer coupled to a RapidFire model 365 to specifically capture and measure KAT2 acetyltransferase activity, converting AcCoA (substrate) to CoA (product).D) Rapid Fire Settings1) Pump1: 6 mM octylammonium acetate, flow rate: 1.5 ml/min; Pump2: 25% acetonitrile, 25% acetone, flow rate: 0.8 ml/min; Pump3: 25% acetonitrile, 25% acetone, flow rate: 0.8 ml/min2) Aspirate 600 ms; Load Time 4500 ms; Elute Time 8000 ms; Re-equilibrate 600 ms3) Area under the curve (AUC) for both substrate and product peaks was determined for KAT2 at M.W. 809.6 [Substrate+H]+ and 767.3 [Product+H]+ with a +/−1 Da tolerance, respectively. |
| Affinity data for this assay | |
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