| Assay Method Information | |
| | KAT6A Biochemical Assay |
| Description: | Inhibition of KAT6A enzymatic activity by test compounds was determined using a radiometric 384-well format assay. A 10-point serial dilution of the test compounds were conducted in DMSO and then a volume of 200 nL was transferred into 384-well assay plate by Echo (Labcyte). 10 μL of 2× enzyme solution (5 nM KAT6A (Active Motif) in assay buffer (50 mM Tris-HCl pH 8.0, 50 mM KCl, 0.1 mM EDTA, 5% glycerol, 1 mM DTT)) was dispensed into assay plate except for low control wells, in which 10 μL of assay buffer was transferred. The plate was incubated at room temperature for 15 min before addition of 10 μL of 2×[3H]-acetyl coenzyme A (Ac-CoA) and substrate peptide mix solution (500 nM (KAT6A) [3H]-Ac-CoA (PerkinElmer) and 800 nM (KAT6A) biotinylated H4(1-30) peptide (GL Biochem) in assay buffer) to each well to start reaction. The plate was incubated at room temperature for 60 min (KAT6A), and then the reaction was stopped by adding 10 μL of stop solution (cold Ac-CoA (Cayman) in 1× assay buffer). 25 μL of reaction in each well was transferred to Flashplate (PerkinElmer) and incubated for another 1 hour at room temperature. The plate was read on Microbeta and the inhibition percentage of each compound treated well was calculated based on the equation in h %=(Max−sample)/(Max−Min)*100 where Max is signal from high control wells with enzyme, and Min is signal from low control wells with assay buffer only. |
| Affinity data for this assay | |
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