| Assay Method Information | |
| | KAT6A Biochemical Assay |
| Description: | TR-FRET is homogeneous proximity assay where Europium-labelled anti-acetyl lysine antibody binds to the acetylated substrate labelled with biotin which in turn binds to streptavidin labelled APC fluorescence acceptor. Europium can transfer energy to APC in the complex and the interaction of two dye-labelled binding partners is detected by the energy transfer between a donor and an acceptor dye, and the subsequent light emission by the acceptor dye. KAT6A transfer an acetyl group from acetyl CoA to lysine amino acids of histones/target proteins. Typically, 5 μL of human-KAT6A (MYST domain 507-778 aa) in assay buffer (100 mM Tris HCl (pH 7.8), 15 mM NaCl, 1 mM EDTA, 0.01% Tween-20, 0.02% BSA, 1 mM DTT) is added to 384-well plate containing 5 μL of selected test compound in final 1% DMSO, serially diluted in 1:3 in an 8-10-point titration. The selected compound of the present invention and enzyme are incubated for 30 min at room temperature. Next, 5 μL of substrate mix containing histone H4 peptide and acetyl-CoA in assay buffer is added to the plate. The final concentrations of H4 peptide and acetyl-CoA are 200 nM and 600 nM respectively. Following 30 min reaction at room temperature, 5 μL of detection mix containing Europium labelled anti-acetyl antibody and streptavidin-APC is added to the reaction wells. The plate is further incubated for 45 min at room temperature and is read in TR-FRET mode (Ex:340 nm; Em: 615 nm and 665 nm) on a plate reader. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |