Science Catalog 2012 Life Cell Signaling cAMP-Glo™ Max Assay Product cAMP-Glo™ Max Assay Size Cat.# Price (Fr) 2 plates V1681 486.00 20 plates V1682 2916.00 10 × 20 plates V1683 Pls. Enq. For Research Use Only. Not for Use in Diagnostic Procedures. Description: The cAMP-Glo™ Max Assay is a homogeneous, bioluminescent and high-throughput assay to measure cyclic AMP (cAMP) levels in cells. Compounds that modulate GPCRs coupled with adenylate cyclase typically alter intracellular cAMP levels. The cAMP-Glo™ Max Assay monitors cAMP levels in cells in response to the effect of agonists, antagonists or test compounds on G protein-coupled receptors (GPCRs). The assay is based on the principle that cyclic AMP (cAMP) stimulates protein kinase A (PKA) holoenzyme activity, decreasing available ATP and leading to decreased light production in a coupled luciferase reaction. This improved version combines the lysis and cAMP reaction buffers into the cAMP-Glo™ ONE Buffer. This new format streamlines the protocol and reduces the time needed to complete the assay. The new ONE Buffer is supplied at a 5X concentration, which provides increased flexibility for starting cell culture volumes. The cAMP-Glo™ Max Assay can be performed in 96-, 384- or 1536-well plates. The cells are induced with a test compound for an appropriate period of time to modulate cAMP levels. After induction, cells are lysed, and the cAMP released stimulates protein kinase A in the reagent. The Kinase-Glo® Reagent is then added to terminate the PKA reaction and detect the remaining ATP via a luciferase reaction. Plates are read using a microplate-reading luminometer. The half-life for the luminescent signal is greater than 4 hours allowing ample time to read the plates and eliminates the need for luminometers with reagent injectors. Features: • Fast and Easy to Use: Improved—Lysis and cAMP detection steps combined (cAMP-Glo™ ONE Buffer). ONE Buffer—5X concentration provides better flexibility for starting cell culture volumes. Assay can be completed in approximately 30 minutes. • Excellent Signal-to-Noise Ratios: Best signal:background ratio of all the cAMP assays. Signal:Background >200 (with cAMP), >15 (on cells). Easily scalable to 1536-well plate formats and beyond. • Proven Luminescent Technology: Powered by Ultra-Glo™ Recombinant Luciferase. No interference by fluorescent compounds. Non-radioactive. Storage Conditions: Store the system at –20°C. Before use, completely thaw all components at room temperature, except for the Protein Kinase A, which should be kept on ice when not at –20°C. After thawing, mix all components thoroughly before use. Once prepared, the cAMP detection solution (cAMPGlo™ ONE Buffer with Protein Kinase A) should not be frozen. Once prepared, the Kinase-Glo® Reagent should be dispensed into aliquots and stored at –20°C. See the product label for the expiration date. Protocol cAMP-Glo™ Max Assay Technical Manual Part# TM347 GloSensor™ cAMP Assay Product GloSensor™ cAMP HEK293 Cell Line pGloSensor™-22F cAMP Plasmid pGloSensor™-20F cAMP Plasmid GloSensor™ cAMP Reagent For Research Use Only. Not for Use in Diagnostic Procedures. Description: The GloSensor™ cAMP Assay presents a novel approach to measuring cAMP levels in live cells. cAMP is a key second messenger involved in signal transduction of GPCRs acting through Gα-s and Gα-i proteins. The new assay is based on the GloSensor™ Technology, a genetically modified form of firefly luciferase into which a cAMP-binding protein moiety has been inserted. Upon binding of cAMP, conformational change is induced leading to increased light output. This live-cell assay excels at kinetic and modulation studies of signaling through cAMP. Researchers can use the GloSensor™ cAMP Assay by transiently expressing a receptor of interest and the biosensor in the cell line of choice. Alternatively, stably transfected cell lines with both the biosensor and the receptor of interest can be made. The protocol is simple: Cells are pre-equilibrated with GloSensor™ cAMP Reagent for approximately 2 hours; then cells are treated with specific agonists/antagonists or compounds, and luminescence is measured after 10–30 minutes. No other reagent additions or manipulations are required. Most any common luminometer with injectors is sufficient to read the assay. GloSensor™ cAMP Reagent is required for use with this assay per the GloSensor™ Limited Use Label License. Choosing the Appropriate Plasmid We offer two variants of the biosensor, and we recommend the pGloSensor™22F cAMP Plasmid as the first choice for most applications. pGloSensor™-22F cAMP Plasmid. Following cell-free expression in vitro, the version encoded by this construct shows an increased EC50 for activation together with increased signal-tobackground ratio at cAMP saturation relative to the version encoded by the pGloSensor™-20F cAMP construct. In general, we have observed similar relationships between the two constructs when their performance is compared in living cells. pGloSensor™-20F cAMP Plasmid. The version encoded by this construct performs well in HEK293 cells at 37°C. Luminescence from the pGloSensor™-22F cAMP Plasmid construct can be more difficult to detect at physiologic temperatures. For a more thorough explanation of the general performance differences between the two plasmids, please consult Section 3.B, Recommendations on Choice of GloSensor™ Plasmid, in the Technical Manual (#TM076). Features: • Best-in-Class Performance: High Z´ and large signal:background ratio values. Ideally suited to HTS/uHTS. Up to 1,000-fold changes in light output obtained. • Live-Cell, Non-Lytic Assay Format: “Zero-step assay” greatly facilitates HTS/uHTS. Easy monitoring of cAMP in live cells enables a more complete analysis of receptor biology. • High Sensitivity and Increased Biological Relevance: Easy detection of low-abundance, endogenous receptors; direct detection of Gi-coupled receptor activation and inverse agonist activity in the absence of added forskolin. PDE inhibitors not needed. Storage Conditions: Store the pGloSensor™ cAMP Plasmid at –20°C and the GloSensor™ cAMP Reagent at –70°C. Store the resuspended GloSensor™ cAMP Reagent at –70°C in single-use aliquots. Protocol GloSensor™ cAMP Assay Technical Manual Part# TM076 Size Cat.# Price (Fr) 2 vials E1261 7953.00 20 µg E2301 796.00 20 µg E1171 796.00 25 mg E1290 536.00 250 mg E1291 2144.00 138 For complete and up-to-date product information visit: www.promega.com/catalog Pagina 141
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