Kinase-Glo Luminescent Kinase Assay, Promega
Supplier: Promega Corporation
The Kinase-Glo Luminescent Kinase Assays are homogeneous non-radioactive systems for determining the activity of purified kinases by quantifying the amount of ATP remaining in solution following a kinase reaction.
- Monitors Activity of Purified Kinases by Quantifying ATP
- Luminescent signal is inversely proportional to the amount of kinase activity
- Three kit formats: Linear to 10microM (Kinase-Glo(R)); 100microM (Kinase-Glo(R) Plus) or 500microM (Kinase-Glo(R) Max) ATP
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.
SignaTECT® Protein Kinase Assays contain the SAM2 biotin capture membrane for a significant improvement over other radioactive protein kinase assays. The membranes are coated with streptavidin for high binding and high specificity. This provides lower backgrounds and higher signal-to-noise ratios than the traditional P81 phosphocellulose method of capture and measurement. The membrane is perforated and numbered for up to 96 kinase reactions. Each system contains all the necessary reaction components. The researcher must supply [[gamma]-32P]ATP.
The filter contains a high density of streptavidin on the matrix. The membranes can linearly bind biotinylated substrates up to the nmol/cm2 range, making them useful for kinetic studies. The membranes are compatible with enzyme assays using radioactive or chemiluminescence detection techniques. It’s not currently recommended for fluorescence techniques. Membranes are available in a partially cut sheet that allows easy separation into 96 individual squares, in an uncut sheet that may be used as a whole membrane or cut to the size desired, or in a 96-well plate that allows washes to be performed with a vacuum manifold or a commercially available plate washer. ProFluor® Assays measure kinase or phosphatase activity with minimal test compound interference. The substrates used in these assays are labeled with rhodamine 110 and produce fluorescent signals higher than the fluorescent signal given off by the test compounds. This allows the fluorescence to be directly correlated with the phosphatase activity. The control peptide included in the assays monitors protease activity to reduce false hits and allows researchers to achieve predicative results with Z' values consistently greater than 0.7 for 96 or 384-well plate formats.
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