Cell Signaling ADP-Glo™ Max Assay Product ADP-Glo™ Max Assay For Research Use Only. Not for Use in Diagnostic Procedures. Description: The ADP-Glo™ Max Assay is a luminescent ADP detection assay that provides a universal, homogeneous, high-throughput screening method to measure ATPase or kinase activity by quantifying the amount of ADP produced in a reaction. The assay can be used to monitor the activity of virtually any ADP-generating enzyme (e.g., kinase or ATPase) when higher ATP concentration is required (up to 5mM). The ADP-Glo™ Max Assay produces a strong signal that positively correlates with enzyme activity and can be adapted to a multitude of plate formats. The assay is performed in two steps: first, after the completion of the ADPproducing reaction, an equal volume of ADP-Glo™ Reagent is added to terminate the reaction and deplete the remaining ATP. Second, the ADP-Glo™ Max Detection Reagent is added to simultaneously convert ADP to ATP, and the latter is converted to light in a coupled reaction with luciferase/luciferin. The ADP-Glo™ Max Assay has a high dynamic range and produces a strong signal at low ATP to ADP conversion, making it well suited for screening lowactivity ATPases such as drug membrane transporters and heat shock proteins. The assay produces minimal false hits and Z´ values of greater than 0.7. Several Kinase Enzyme Systems are available. Visit www.promega.com/kinase/ to see the collection. Features: • High Signal Strength at Low ATP Conversion: Users can measure enzyme activity that more closely mimics physiological conditions. This makes the assay very well suited for low-activity ATPases/kinases. • Sensitive: The assay is sensitive to low concentrations of ADP, thus requiring less enzyme than other assays; cost savings. • Universal: The assay can be used with virtually with any ADP-producing enzyme—enables researchers to screen a wider range of enzymes using a single platform. • Accommodate Wide Range of ATP Levels: The assay can be used at ATP concentrations up to 5mM, important for enzymes with high Km values for ATP and for mode of action studies. • Accurate: Accurately measures ADP levels at a wide range of starting ATP concentrations; users assured that activity measured truly reflects enzyme activity and produces accurate IC50s comparable to radioactivity-based assays. Storage Conditions: Store the system at –20°C. Before use, thaw all components completely at room temperature. Once thawed, mix all components thoroughly before use. Because ATP is naturally prone to hydrolysis after freeze-thaw cycles dispense into single-use aliquots and store at –20°C. Once prepared, dispense, ADP-Glo™ Max Detection Reagent (ADP-Glo™ Max Detection Buffer + Substrate) into aliquots and store at –20°C. ADP-Glo™ Max Detection Buffer may form a precipitate when thawed. See Section 3.A of the Technical Manual for a protocol to dissolve any precipitate. For convenience, ADP-Glo™ Reagent and ADP-Glo™ Max Detection Reagent may be kept at room temperature (22°C) for 24 hours without loss of signal. Protocol ADP-Glo™ Max Assay Technical Manual Part# TM343 ATP ADP Luminescence Principle of the ADP-Glo™ Max Assay. The assay is performed in two steps: 1) After the ATPase or kinase reaction, ADP-Glo™ Reagent is added to terminate the reaction and deplete the remaining ATP; and 2) the ADP-Glo™ Max Detection Reagent is added to convert ADP to ATP and allow the newly synthesized ATP to be measured using a luciferase/luciferin reaction. The light generated correlates to ADP present and ATPase activity. Size Cat.# Price (Fr) 1,000 assays V7001 891.00 10,000 assays V7002 4455.00 ATPase Reaction Kinase or 3 Step 1: ATP Depletion ADP-Glo™ Reagent Step 2: ADP Detection ADP-Glo™ Max Detection Reagent For complete and up-to-date product information visit: www.promega.com/catalog 55 Cell Signaling 8051MB Pagina 58
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