Kinase-Glo Luminescent Kinase Assay
The Kinase-Glo Luminescent Kinase Assays are homogeneous non-radioactive methods for determining the activity of purified kinases by quantifying the amount of ATP remaining in solution following a kinase reaction. The assays are designed for use with multiwell plate formats, making them ideal for automated high-throughput screening (HTS), and they can be used to assay protein, lipid and sugar kinases. The assay procedure involves addition of a single reagent directly to a completed kinase reaction. This addition results in the generation of a luminescent signal correlated with the amount of ATP present and inversely proportional to the amount of kinase activity. The Kinase-Glo Assays generate a glow-type luminescent signal produced using a patented stabilized luciferase (Ultra-Glo Luciferase) coupled with a proprietary buffer system. When assayed in the presence of kinase reaction buffers, such as the reaction buffer for PKA, the half-life of the luminescent output is greater than five hours, eliminating the need for luminometers with injectors and allowing for batch plate processing. The assay produces excellent Z'-factor values of greater than 0.7 in 96- and 384-well formats, easily detects known kinase inhibitors and provides IC50 values comparable to those reported in the literature. The Kinase-Glo Assay systems are differentiated by their linear response to ATP (see figure below). The original Kinase-Glo Assay is linear to 10uM ATP, while Kinase-Glo Plus Assay is linear to 100uM ATP. The newest assay, Kinase-Glo Max, is linear to 500uM ATP, making it well suited for use with kinases with high Km for ATP as well as for screening for kinase inhibitors that do not compete at the ATP binding site.