| Assay Method Information | |
| | KAT6A and KAT6B Biochemical Assay |
| Description: | Testing for the inhibition activities of various compounds disclosed herein was carried out at room temperature in assay buffer containing 100 mM Tris/HCl Ph7.8, 15 mM NaCl, 0.01% chicken egg white albumin, 1 mM DTT and 0.01% Tween-20. Compounds in DMSO were dispensed into wells of a black, 384-well plate (Corning 4514) using ECHO555 (Beckman). The final concentration ranges of the test compounds were 0.0625-1,000 nM, 0.625-10,000 nM, or 0.313-5,000 nM. Five μL of KAT6A or KAT6B enzyme solution were added to wells, and the plate was incubated for 1 hour at room temperature (rt). After incubation for 1 hour at rt, 5 μL H4 and AcCoA substrate solutions were added to the wells to initiate reaction, and the plate was incubated. After 2 hours reaction, 5 μL detection reagent were added to the wells, and the plate was incubated for 2 hours. After two hours incubation, detection reagent containing 5 nM Anti-histone H4 antibody (ABCAM #ab177790), 100 μM formic acid (sigma #06473-100ML), 0.1 test/μL streptavidin-XL665 (PerkinElmer #610SAXLG), 0.05 test/μL PAb anti-rabbit IgG-Eu (PerkinElmer μ61PARKLA) was added to the wells. TR-FRET was measured on a microplate reader (PHERAstar FSX, BMG labtech). |
| Affinity data for this assay | |
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