Kinase-Glo® Luminescent Kinase Assays

Description

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 K_m 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.
• Screen for Non-ATP Binding Site Inhibitors: Use ATP concentrations as high as 500μM (Kinase-Glo® Max Assay).

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