Cell Signaling ® Kinase-Glo Product Kinase-Glo ® Luminescent Kinase Assays Size Cat.# Luminescent Kinase Assay 10 ml V6711 10 × 10 ml V6712 100 ml V6713 Kinase-Glo ® Max Luminescent Kinase Assay 10 × 100 ml V6714 10 ml V6071 10 × 10 ml V6072 100 ml V6073 Kinase-Glo ® Plus Luminescent Kinase Assay 10 × 100 ml V6074 10 ml V3771 10 × 10 ml V3772 100 ml V3773 10 × 100 ml V3774 For Research Use Only. Not for Use in Diagnostic Procedures. Description: The Kinase-Glo® A. Price (Fr) 156.00 858.00 773.00 Pls. Enq. 172.00 946.00 852.00 Pls. Enq. 164.00 902.00 812.00 Pls. Enq. Luminescent Kinase Assays are homogeneous nonradioactive 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). The assay procedure involves a single reagent addition 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® to ATP (see figure below). The original Kinase-Glo ATP, while Kinase-Glo® Kinase-Glo Features: • Assay a Variety of Kinases: Use with a wide range of kinases (including lipid, sugar and alcohol kinases) and substrates (peptides, proteins, lipids, sugars and alcohols). • Obtain Reliable Results: Less interference from library compounds. 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/myway/ • Screen for Non-ATP Binding Site Inhibitors: Use ATP concentrations as high as 500μM (Kinase-Glo® Max Assay). .Storage Conditions: Store at –20°C. Avoid multiple freeze-thaw cycles and exposure to frequent temperature changes. These fluctuations can greatly alter product stability. Protocol Kinase-Glo ® Luminescent Kinase Assay Platform Technical Bulletin Part# TB372 Assay systems are differentiated by their linear response ® Assay is linear to 10μM Plus Assay is linear to 100μM ATP. The newest assay, ® 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. B. 1.6 × 108 1.4 × 108 1.2 × 108 1 × 108 8 × 107 6 × 107 4 × 107 2 × 107 0 0 y = 280837x + 2 × 1016 r2 = 0.9971 3.0 × 108 2.5 × 108 2.0 × 108 1.5 × 108 1.0 × 108 0.5 × 108 0 r2 =0.9993 0 20 40 60 ATP (µM) 80 100 120 3 100 200 300 400 ATP (µM) Kinase-Glo Panel A. Kinase-Glo® Plus Assay. Panel B. Kinase-Glo® ® Assay is linear to 10μM (data not shown). ProFluor Product ProFluor ® ® PKA Assay Size PKA Assay For Research Use Only. Not for Use in Diagnostic Procedures. Description: The ProFluor® 4 plate 8 plate Cat.# V1240 V1241 Price (Fr) 1304.00 2348.00 PKA Assay measures protein kinase A activity using purified kinase in a multiwell plate format and involves “add-mix-read” steps only—ideal for high-throughput applications. The assay begins with a standard kinase reaction performed with a provided PKA bisamide rhodamine 110 peptide substrate. Following the kinase reaction, a termination buffer containing a protease reagent is added, which simultaneously stops the kinase reaction and removes amino acids specifically from the nonphosphorylated PKA substrate, liberating highly fluorescent rhodamine 110. Phosphorylated PKA substrate, however, is resistant to digestion by the protease reagent and remains nonfluorescent. Thus, fluorescence intensity measured in this assay is inversely correlated with kinase activity. The assay produces excellent Z´-factor values (>0.8) in either 96- or 384-well plate formats and easily distinguishes known PKA inhibitors from other compounds. Features: • Achieve Highly Predictive Results: Robust Z´ values greater than 0.7 in either 96- or 384-well plate formats. • Observe Minimal Test Compound Interference: Fluorescent signal from Rhodamine 110 is much stronger than fluorescent signals given off by test compounds. • Homogeneous: Add-mix-read format reduces the number of steps. • Non-Radioactive: No radioactive waste disposal and safety issues. • Choose Your Configuration: Learn more about our custom options for this product at: www.promega.com/myway/ Storage Conditions: Store the entire system at –20°C. Protect the PKA R110 Substrate from light. For best results, make solutions fresh and use immediately. System components should be thawed on ice and returned to –20°C as soon as possible. The PKA R110 Substrate is provided in 100% DMSO and therefore requires thawing at room temperature. Protocol ProFluor ® PKA Assay Technical Bulletin For complete and up-to-date product information visit: www.promega.com/catalog Part# TB315 61 Luminescent output correlates with amount of ATP. A direct relationship exists between the luminescence measured with the Kinase-Glo 500 600 ® Assay systems and the amount of ATP in the reaction. Max Assay. The Cell Signaling Luminescence (RLU) Luminescence (RLU) 6979MB Pagina 64
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