| Assay Method Information | |
| | KAT8 Assay Protocol |
| Description: | 1. Final reaction conditions are 30 nM KAT8 Full length enzyme, 1 μM AcCoA, 2 μM H3 1-21 peptide, in a 20 μL reaction volume.2. Add 200 nL of diluted compound to the assay plate (384-well microtiter V-bottom polypropylene plates [Greiner Inc]) or 200 nL of DMSO or 200 nL reference inhibitor for the control wells3. Add 10 μL of KAT8 enzyme mix to the assay plate4. Add 10 μL of H3 1-21 peptide and AcCoA mix to the assay plate5. Terminate the reaction after 60 mins with the addition of 2 μL of 5% Formic Acid6. Neutralize the terminated acidified reaction for 30 mins with 2.0 μL of 10% Sodium Bicarbonate (1% final) [Sigma Inc]7. Transfer 2.6 μL of the neutralized reaction to a 384-well SAMDI plate [SAMDI Tech] surface containing the proprietary Biotin-NeutrAvidin monolayer (Mrksich M. Mass spectrometry of self-assembled monolayers: a new tool for molecular surface science. ACS Nano. 2008 January;2(1):7-18. doi: 10.1021/nn7004156. PMID: 19206542; PMCID: PMC2600870 and Michael D. Scholle, Patrick T. O'Kane, Sandra Dib, Zachary A. Gurard-Levin, Label-free duplex SAMDI-MS screen reveals novel SARS-CoV-2 3CLpro inhibitors, Antiviral Research, Volume 200, 2022, 105279, ISSN 0166-3542.)8. Immobilize the reactions on the SAMDI plate surface in a humidified chamber for 45 mins at rt9. Wash the SAMDI plate surface 5 times using purified Milli-Q H2O and allow to dry completely with pressurized air using air blades10. Apply α-Cyano-4-hydroxycinnamic acid [Sigma Inc] matrix using SAMDI proprietary method11. Measure enzyme activity using a MALDI TOF/TOF System [AB Sciex] and determine the area under the curve for the substrate and product peaks at MW2723 [Substrate+H]+ and 2765 [Product+H]+ with a ±1 Da tolerance, respectively |
| Affinity data for this assay | |
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