| Assay Method Information | |
| | Inhibition of KAT6A Enzyme Activity Assay |
| Description: | 1× experimental buffer (modified Tris buffer) was prepared, and the compounds were dissolved with 100% DMSO to form 10 mM reservoir solution and diluted according to a certain concentration gradient. The compounds were transferred to a 384-well plate using an automatic sampler Echo, and the final concentration of DMSO was 1%. 1× KAT6A enzyme (catalytic domain) solution was prepared. A mixed solution of [3H]-acetyl-CoA (PERKIN ELMER, Cat. No. NET290250UC) and the substrate peptide H3 (1-21) was prepared. 10 μL of the enzyme solution was transferred to the 384-well plate and incubated at room temperature for 15 minutes. 10 μL of the mixed solution of [3H]-acetyl-CoA and the substrate peptide H3 (1-21) were added to the plate to initiate the reaction. The plate was incubated at room temperature for 1 hour. 10 μL of the stop solution was added to terminate the reaction. 25 μL of the reaction solution was transferred to a Flashplate (Perkin Elmer, Cat. No. SMP410A001PK) and incubated at room temperature for 1 hour. The plate was read using Microbeta. The inhibition rate of the compounds was calculated using the formula of Excel: inhibition %=(Max-Signal)/(Max−Min)*100%. Where max is the value of the DMSO control, min is the value of the non-enzymatic control, and signal is the value of the well where the compound was tested. |
| Affinity data for this assay | |
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