GloSensor™ Technology

Luciferase Biosensor for Real-Time Detection of Signaling Events

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.

GloSensor™ luciferase is circularly permuted, joining the wild-type N- and C-termini with a polypeptide or protein domain and creating engineered termini elsewhere within the structure. Analyte binding or protease cleavage leads to a conformational shift in the biosensor that promotes large increases in luminescence activity. The magnitude of the luminescence increase is directly proportional to the amount of analyte or protease activity present.

Simple, Scalable, Live-Cell Protocol

GloMax Discover System Multimode Plate Reader

The live-cell, non-lytic assay protocol is easy to perform, allowing biosensor activity to be monitored continuously or at a single time point. The kinetic profile of cell signaling events can be followed in real time and the assays easily scale to high-density microplate formats, including 96-, 384- and 1536-well plates.

The protocol steps are:

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.

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.

Order Additional cAMP Vectors and Cell Lines

12766MA_600px — **Kinetic response of GloSensor™ cAMP compared to a cAMP immunoassay.** HEK293 cells transiently expressing the pGloSensor™-22F cAMP Plasmid were treated sequentially with 1µM isoproterenol (ISO), 10µM propranolol (PRO), and 10µM forskolin (FSK). The live-cell, nonlytic GloSensor™ cAMP Assay protocol was used to monitor cAMP levels, whereas replicate wells were lysed at each time point for the cAMP immunoassay (Enzo Life Sciences). Samples above approximately 20nM required dilution for the cAMP immunoassay to be within the linear range of the standard curve. (ISO = ADRB2 agonist, PRO = ADRB2 antagonist, FSK = cell permeable activator of adenylyl cyclase).

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.

Order cGMP Assay

12767MA_600px — **Real-time cGMP monitoring.** HEK293 cells transiently expressing the 42F construct were treated with varying doses of sodium nitroprusside (SNP), a nitric oxide donor, to activate the endogenous soluble guanylyl cyclase (sGC). The live-cell, non-lytic GloSensor™ cAMP Assay protocol was used to monitor the cGMP response in real-time.

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 enabling a method for in vivo measure of apoptosis in animal models. 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). You can read more about this work in this work in the article: A real-time assay of caspase-3/7 activity in cell culture and animal models.

The GloSensor™ caspase 3/7 biosensor plasmid, stable cells lines and granzyme biosensor plasmids are available as custom assay materials. These biosensor plasmids can be used directly or modified by the insertion of novel cleavage sites to create biosensors for a particular protease of interest.

Order Protease Assay

GloSensor™ Plasmids

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-to-background 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 thorough explanation of the performance differences between the two plasmids, please consult Section 3.B, Recommendations on Choice of GloSensor™ Plasmid, in Technical Manual #TM076 (provided in the Protocol Section below).

pGloSensor-22F cAMP Plasmid. — pGloSensor™-20F cAMP, Accession Number EU770615; Sequence (.txt file)
pGloSensor™-22F cAMP, Accession Number GU174434; Sequence (.txt file)

Protocols

Complete Protocol

GloSensor™ cAMP Assay Technical ManualPDF (903 KB) – English

Quick Protocols

GloSensor cAMP Assay Quick Protocol FB113PDF (286 KB) – English

What's in the box?

Item	Part #	Size
GloSensor™ cAMP Reagent	E129A	1 × 25mg

SDS

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Certificate of Analysis

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Use Restrictions

For Research Use Only. Not for Use in Diagnostic Procedures.

Storage Conditions

See Protocol for detailed storage recommendations.

What's in the box?

Item	Part #	Size
GloSensor™ cAMP Reagent	E129B	1 × 250mg

SDS

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Certificate of Analysis

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Use Restrictions

For Research Use Only. Not for Use in Diagnostic Procedures.

Storage Conditions

See Protocol for detailed storage recommendations.

What's in the box?

Item	Part #	Size
pGloSensor™-22F cAMP Plasmid	E230A	1 × 20μg

SDS

Search for SDS

Certificate of Analysis

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Use Restrictions

For Research Use Only. Not for Use in Diagnostic Procedures.

Storage Conditions

See Protocol for detailed storage recommendations.

Patents and Disclaimers

BY USE OF THIS PRODUCT, RESEARCHER AGREES TO BE BOUND BY THE TERMS OF THIS LIMITED USE LABEL LICENSE. If researcher is not willing to accept the terms of this label license, and the product is unused, Promega will accept return of the unused product and provide researcher with a full refund.
Researchers may use this product in their own research solely for the luminescent determination of molecular binding, enzymatic activity or other functional activity. Researchers shall have no right to modify or otherwise create variations of the nucleotide sequence encoding the GloSensor™ technology of this product (i.e., the bioluminescent chimeric protein comprising luciferase and other functional domains). Researchers may transfer derivatives to others provided that recipients agree to be bound by the terms of this label license prior to such transfer. No other use or transfer of this product is authorized without the prior express written consent of Promega. In addition, researchers must either: (1) use GloSensor™ cAMP Reagent for in vitro and in cell applications, and VivoGlo™ Luciferin, In Vivo Grade, for animal applications for all determinations of luminescence activity of this product and derivatives; or (2) contact Promega to obtain a license for use of the product and derivatives in conjunction with luminescent assay reagents not purchased from Promega. PROMEGA MAKES NO REPRESENTATIONS OR WARRANTIES OF ANY KIND, EITHER EXPRESSED OR IMPLIED, INCLUDING FOR MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE WITH REGARDS TO THE PRODUCT. The terms of this label license shall be governed under the laws of the State of Wisconsin, USA.

Patent Pending.

U.S. Pat. No. 8,008,006.

U.S. Pat. No. 7,728,118 and other patents pending.

What's in the box?

Item	Part #	Size
pGloSensor™-20F cAMP Plasmid	E117A	1 × 20μg

SDS

Search for SDS

Certificate of Analysis

Search by lot number

Use Restrictions

For Research Use Only. Not for Use in Diagnostic Procedures.

Storage Conditions

See Protocol for detailed storage recommendations.

Patents and Disclaimers

BY USE OF THIS PRODUCT, RESEARCHER AGREES TO BE BOUND BY THE TERMS OF THIS LIMITED USE LABEL LICENSE. If researcher is not willing to accept the terms of this label license, and the product is unused, Promega will accept return of the unused product and provide researcher with a full refund.
Researchers may use this product in their own research solely for the luminescent determination of molecular binding, enzymatic activity or other functional activity. Researchers shall have no right to modify or otherwise create variations of the nucleotide sequence encoding the GloSensor™ technology of this product (i.e., the bioluminescent chimeric protein comprising luciferase and other functional domains). Researchers may transfer derivatives to others provided that recipients agree to be bound by the terms of this label license prior to such transfer. No other use or transfer of this product is authorized without the prior express written consent of Promega. In addition, researchers must either: (1) use GloSensor™ cAMP Reagent for in vitro and in cell applications, and VivoGlo™ Luciferin, In Vivo Grade, for animal applications for all determinations of luminescence activity of this product and derivatives; or (2) contact Promega to obtain a license for use of the product and derivatives in conjunction with luminescent assay reagents not purchased from Promega. PROMEGA MAKES NO REPRESENTATIONS OR WARRANTIES OF ANY KIND, EITHER EXPRESSED OR IMPLIED, INCLUDING FOR MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE WITH REGARDS TO THE PRODUCT. The terms of this label license shall be governed under the laws of the State of Wisconsin, USA.

Patent Pending.

U.S. Pat. No. 8,008,006.

U.S. Pat. No. 7,728,118 and other patents pending.

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Citations

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GloSensor™ Technology

Live-Cell Biosensors for cAMP, cGMP or Protease Activity

Luciferase Biosensor for Real-Time Detection of Signaling Events

Simple, Scalable, Live-Cell Protocol

Perform cAMP, cGMP or Protease Assays

Monitor Intracellular cAMP in a Live-Cell Assay

Monitor Intracellular cGMP in a Live-Cell Assay

Luminescent Detection of Protease Activity

GloSensor™ Plasmids

Protocols

Complete Protocol

Quick Protocols

Specifications

What's in the box?

SDS

Certificate of Analysis

Use Restrictions

Storage Conditions

Specifications

What's in the box?

SDS

Certificate of Analysis

Use Restrictions

Storage Conditions

Specifications

What's in the box?

SDS

Certificate of Analysis

Use Restrictions

Storage Conditions

Patents and Disclaimers

Specifications

What's in the box?

SDS

Certificate of Analysis

Use Restrictions

Storage Conditions

Patents and Disclaimers

Resources

Citations

Similar Products

cAMP-Glo™ Max Assay

cAMP-Glo™ Assay

Frequently Used With

FuGENE® HD Transfection Reagent

GloMax® Discover System

ViaFect™ Transfection Reagent

PureYield™ Plasmid Miniprep System

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