What is GloSensor?
What is GloSensor?
The GloSensor™ cAMP Assay provides an extremely sensitive and easy-to-use format for the interrogation of overexpressed or endogenous GPCRs that signal via changes in the intracellular concentration of cAMP. Upon binding to cAMP, conformational changes occur that promote large increases in light output (1–4).
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 measured?
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 does forskolin increase cAMP?
Forskolin increases intracellular cAMP level through activation of adenylyl cyclase in various cell types. In this study, the authors find that forskolin-induced increases in cAMP levels enhance mAb production in recombinant CHO cell cultures.
What are cAMP levels?
Cyclic AMP (cAMP) is an intracellular second messenger to a wide variety of hormones and neurotransmitters. In T cells, elevated cAMP levels antagonize T cell activation by inhibiting T cell proliferation and by suppressing the production of IL-2 and IFN-γ.
What is a cAMP assay?
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. The cells are induced with a test compound for an appropriate period of time to modulate cAMP levels.
What is the biological source of forskolin?
Forskolin (coleonol) is a labdane diterpene that is produced by the Indian Coleus plant (Plectranthus barbatus).
Is cAMP a neurotransmitter?
Cyclic AMP, Adenylyl Cyclases, and Phosphodiesterases Cyclic AMP (cAMP) is an intracellular second messenger to a wide variety of hormones and neurotransmitters.
Why is it called cyclic?
In a finite-size system this situation usually corresponds to the rotational degree of freedom, that’s why it is called “cyclic”.
Is GPCR an enzyme?
What Second Messengers Do GPCR Signals Trigger in Cells? This membrane-associated enzyme catalyzes the synthesis of not one, but two second messengers — DAG and IP3 — from the membrane lipid phosphatidyl inositol. This particular pathway is critical to a wide variety of human bodily processes.
What is a cAMP accumulation assay?
5 cAMP accumulation assay. Measurement of cAMP release is the assay of choice for Gα13 protein-coupled receptors. Gs directly stimulates cAMP release through activation of adenylyl cyclase. The lysate was used to measure cAMP concentration by a competitive protein-binding assay using [8-3H]cAMP.
How is glosensor technology used in live cells?
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.
What can glosensor 3 / 7 biosensor be used for?
The GloSensor™ caspase 3/7 biosensor, together with a similar construct designed to detect granzyme B, have also been used for the detection of target cell apoptosis mediated by in vitro-derived or ex vivo cytotoxic T lymphocytes (CTLs).
What can the glosensor cAMP assay be used for?
The GloSensor™ cAMP Assay can be used to monitor activation of endogenous or overexpressed Gs- or Gi-coupled 7-TM, G-protein coupled receptors (GPCRs), together with phosphodiesterases (PDEs) that are responsible for degrading intracellular cAMP.
What is the glosensor 30F plasmid used for?
A novel GloSensor™ protease construct, the 30F plasmid, was developed for live-cell, non-lytic assays of protease activity for use in both cell culture and in vivo models.
