What is GloSensor?
GloSensor™ technology provides a platform of flexible luciferase-based biosensors for real-time detection of signaling events in live cells with sensitivity, linearity and specificity.
How does the GloSensor cAMP assay work?
GloSensor™ cAMP Assay Technical Manual The assay uses genetically encoded biosensor variants with cAMP binding domains fused to mutant forms of Photinus pyralis luciferase. Upon binding to cAMP, conformational changes occur that promote large increases in light output.
How are cAMP levels determined?
The cAMP-Glo™ Max Assay measures cAMP levels through protein kinase A (PKA), which is activated upon release of its regulatory subunits after binding to cAMP (Figure 1). PKA is a component of the cAMP Detection Solution, and once activated, PKA will use any ATP present to phosphorylate its substrate.
How is cGMP measured?
The method of choice for measuring tissue levels of cGMP is a radioimmunoassay, but it can also be measured using a scintillation proximity assay.
How is cyclic AMP measured?
(a) Cyclic nucleotides can be monitored by directly measuring cyclic nucleotide gated channel (CNGC) currents or calcium influx through these channels by a calcium sensitive dye; (b) FRET sensors can be constructed based on the fluorophore-labeled protein kinase A (PKA) heterotetramer (R and C subunits), partially …
Do all GPCRs have 7 transmembrane domains?
GPCRs are integral membrane proteins that possess seven membrane-spanning domains or transmembrane helices. The extracellular parts of the receptor can be glycosylated. These extracellular loops also contain two highly conserved cysteine residues that form disulfide bonds to stabilize the receptor structure.
How do GPCRs activate G proteins?
As their name implies, GPCRs interact with G proteins in the plasma membrane. When an external signaling molecule binds to a GPCR, it causes a conformational change in the GPCR. This change then triggers the interaction between the GPCR and a nearby G protein.
How Cyclic AMP causes vasodilation?
Cyclic GMP (cGMP) mediates the relaxing action of a variety of vasodilator drugs and endogenous vasodilator substances. Cyclic AMP (cAMP) mediates relaxation by beta-adrenergic agonists as well as other activators of adenylate cyclase.
What is a G protein coupled 7 transmembrane receptor?
G protein-coupled receptors, or GPCRs, also known as 7-Transmembrane receptors (7-TM receptors), are integral membrane proteins that contain seven membrane-spanning helices. Upon ligand binding, the GPCR undergoes a conformational change which is transmitted to the G protein causing activation.
What does the glosensor™ cGMP assay measure?
The GloSensor™ cGMP Assay is used to monitor changes in the intracellular concentration of the second messenger cGMP.
How sensitive is the glosensor 22F?
The 22F construct is sensitive enough to monitor inverse agonist activity or Gi-coupled receptor activation in the absence of added forskolin, but possess a large enough dynamic range to monitor very large increases in intracellular cAMP concentration. Kinetic response of GloSensor™ cAMP compared to a cAMP immunoassay.
How to use glosensor™ camp reagent for luminescence?
Pre-equilibrate cells with GloSensor™ cAMP Reagent for ~2 hours. Treat with specific agonists/antagonists or library compounds. Measure luminescence after 10–30 minutes either continuously or at a single time point. Any luminometer with injectors is sufficient to read the assay.
Which pglosensor™-22F camp plasmid is best for my application?
The sequence for each differs, giving biosensors with a range of affinities for cAMP. In general, the pGloSensor™-22F cAMP Plasmid is best-suited for most applications, providing a unique combination of sensitivity and dynamic range.