Enzyme and luciferase methods

A standard 20 μl reaction buffer contained: 50 mM Tris–HCl, pH 8.5, 50 mM MgCl₂, 1 mM Dithiothreitol (DTT), 100 μM DIAL nucleotide, and varied amounts of ITPA. After 1 h incubation at 37°C, 5 μl of this reaction solution was added to 20 μl Kinase-Glo Luminescent Kinase Assay reaction solution (prepared as instructed for ATP determination kit) in a 96-well plate. The bioluminescence signal was recorded at 1 min intervals over 1 h by microplate fluorometer.