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 G-alpha-s and G-alpha-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. 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 S/B 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 degrees C. Luminescence from the pGloSensor-22F cAMP Plasmid construct can be more difficult to detect at physiologic temperatures. Therefore, we recommend the pGloSensor-22F cAMP Plasmid as the first choice for most applications. 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).|
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.