Description
Perform cAMP, cGMP or Protease Assays
Monitor Intracellular cAMP in a Live-Cell Assay
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. Intracellular changes in cAMP concentration can be followed using a live-cell, non-lytic assay format, allowing real-time monitoring of changes in cAMP concentration.
Different biosensors are available for use in the GloSensor™ cAMP Assay. In each case, a cAMP binding domain is fused to a circularly permuted form of firefly luciferase. 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. 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.
Monitor Intracellular cGMP in a Live-Cell Assay
The GloSensor™ cGMP Assay is used to monitor changes in the intracellular concentration of the second messenger cGMP. The assay can be used to monitor activation of endogenous or overexpressed guanylyl cyclases, including soluble guanylyl cyclase (sGC) or atrial natriuretic peptide receptor (NPRA and NPRB) isoforms, together with the phosphodiesterases (PDEs) that are responsible for degrading intracellular cGMP. Intracellular changes in cGMP concentration can be followed using a live-cell, non-lytic assay format, allowing real-time monitoring of changes in cGMP concentration.
Different biosensors are available for use in the GloSensor™ cGMP Assay. In each case, a cGMP binding domain is fused to a circularly permuted form of firefly luciferase. The cGMP binding domain and sequence for each differs, giving biosensors with different affinities for cGMP. In general, the 40F construct is preferred when overexpressing a guanylyl cyclase, providing maximal dynamic range at high intracellular levels of cGMP. In contrast, the 42F construct is preferred when assaying an endogenous guanylyl cyclase, providing maximal sensitivity for lower intracellular levels of cGMP.
Luminescent Detection of Protease Activity
The GloSensor™ Protease Assay is used to monitor protease activity both in vitro and in vivo. The approach utilizes circularly permuted forms of luciferase where the wild-type N- and C-termini are tethered with a peptide containing a protease cleavage site. Once cleaved by the corresponding protease, a conformational constraint is lifted, resulting in large increases in luminescence activity. Peptides containing protease cleavage sites up to 20 amino acids in length have been validated in vitro, using this technology, including proteases that require P’ residues for efficient cleavage. 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. When engineered with a caspase-3/7 cleavage site, the GloSensor™ caspase 3/7 biosensor enables a sensitive, real-time approach for the detection of caspase-3/7 activity, providing a method for in vivo measure of apoptosis in animal models. You can read more about this work in the article: A real-time assay of caspase-3/7 activity in cell culture and animal models. Similar biosensor constructs designed to detect granzyme B and viral proteases have been reported in the literature. The GloSensor™ caspase 3/7 biosensor plasmid is available through our partner Addgene , which can be used directly or modified by inserting novel cleavage sites to create biosensors for a particular protease of interest. Custom GloSensor™ protease construct or stable cell line development can also be requested through our Tailored R&D Solutions Team .
