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 10μM ATP, while Kinase-Glo® Plus Assay is linear to 100μM ATP. The newest assay, Kinase-Glo® Max, is linear to 500μM 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.
Features - Benefits
Assay a Variety of Kinases: Can be used for a wide range of kinases (including lipid, sugar and alcohol kinases) and substrates (peptides, proteins, lipids, sugars and alcohols). Obtain Reliable Results: Luminescence is much less susceptible to interference from library compounds than other luciferase-based ATP detection reagents. Z´-factor values greater than 0.7 in either 96- or 384-well plate formats. Simplify Your Assay: Homogeneous—everything is performed in a single well. Non-Radioactive: No radioactive waste disposal and safety issues. Automate This Assay: Validated automated methods available at: www.promega.com/automethods/ Choose Your Configuration: Learn more about our custom options for this product at: www.promega.com/custom/ Screen for Non-ATP Binding Site Inhibitors: Use ATP concentrations as high as 500μM (Kinase-Glo® Max Assay).
Applications
For more information, see the Protocols & Applications Guide.
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