Targeting the cGAS-STING Pathway Using a Homogenous, HTS Compatible Transcreener cGAS Assay

Transcreener cGAMP Assay Kit (TR-FRET) Method

Ordering Information

Instrument Compatibility

Related Resources

Transcreener cGAMP Assay Kit - TR-FRET Readout

The Transcreener® cGAMP Assay with TR-FRET readout uses displacement of a tracer by cGAMP to measure enzymatic activity. This disrupts FRET from the antibody-bound Terbium chelate leading to a decrease in the TR-FRET signal. In addition to the time-resolved signal, the assay uses a far-red tracer to minimize interference from fluorescent compounds and light scattering.

The assay produces excellent data quality (Z' ≥ 0.7) at cGAMP ranges between 0.5 μM and 100 μM. Both reagents & signal are stable at room temperature for over 24 hours, providing outstanding flexibility for automated high-volume screening programs. The universal detection method allows screening of virtually any cGAMP-producing enzyme in a high throughput format.

Used in over millions of wells by pharma since 2006, Transcreener is an extensively validated enzyme detection method backed by the best technical support in the industry.

This assay is designed to be used with purified enzyme preparations. Transcreener assays are not validated with cell lysates, blood, serum, or other biological samples.

Direct Detection of cGAMP to Measure cGAS Enzymatic Activity

Time-Resolved Förster Resonance Energy Transfer (TR-FRET)

cGAMP Assay Kit Schematic - FRET Readout

Learn More About the Transcreener cGAMP Assay

Transcreener cGAMP TR-FRET Assay Tech Manual

Applications

Measure enzymatic activity of cGAS
Screen compound libraries for cGAS inhibitors
Quantify inhibitor potency
Determine inhibitor-cGAS residence time

Features

Direct detection of unlabeled cGAMP
Easy to use, homogenous, one step format
Robust assay Z' > 0.7 under initial velocity conditions
Far-red fluorescent readouts minimize compound interference
A safe, non-radioactive method

Easy-to-Use, Mix-&-Read, HTS-Ready

Run your enzyme reaction, add Transcreener reagents, and read your plates. The cGAS assay kit is compatible with 96, 384, and 1536-well formats.

Important Messenger in the STING Pathway

DNA in the cytosol of eukaryotic cells indicates infection or cellular damage, and it elicits a robust immune response, driven by type I interferon (IFN) induction. cGAS is an exquisitely sensitive sensor for double-stranded DNA in the cytoplasm. Binding of DNA activates the enzyme to produce a unique cyclic dinucleotide second messenger, cGAMP, which acts as an agonist for the STING (stimulator of interferon genes) receptor, leading to induction of a type I interferon response.

Example cGAMP Titration

Excellent Z' Down to nM cGAMP Levels

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Far-Red TR-FRET Readouts Validated on Major Multimode Readers

Supplier	Instrument	TR-FRET Assays
	TriStar²S LB 942	Validated
	Tristar 5	In Review
	Mithras² LB 943	Validated

	Cytation^TM 5	Validated
	Cytation^TM 3	Validated
	Cytation^TM 1	Validated
	Synergy^TM H1	Validated
	Synergy^TM 2/H4/4	Validated
	Synergy^TM HTX	Not Capable
	Synergy^TM Neo 2	Validated

	POLARstar^® Omega	Validated
	FLUOstar^® Omega	Validated
	PHERAstar^® FSX	Validated
	PHERAstar^® Plus/FS	Validated
	CLARIOstar^® /Plus	Validated
	VANTAstar^TM	Validated

	Sense	In Review

	Analyst^® GT/HT	Validated
	Gemini^® XPS/EM	Not Capable
	SpectraMax^® M2/M2e	Not Capable
	SpectraMax^® M5/M5e/FlexStation^® 3	Validated
	SpectraMax^® Paradigm	Contact Us
	SpectraMax^® iD3/iD5	Validated
	SpectraMax^® i3x	In Review

	EnVision^®/EnVision^® Xcite	Validated

	Infinite^® M1000/M1000Pro/Safire2^TM	Validated
	Infinite^® M200	Not Capable
	Infinite^® F500	Validated
	Infinite^® F200/Ultra^evolution	Contact Us
	Spark^TM 10M	Validated

Validation Criteria for Plate Readers

384-Well Format
Z'-Factor ≥ 0.7 at 10% conversion of 10 μM ATP
Z' specifications to be met using Transcreener ADP² Assay Reagents
Read time to achieve Z' specifications ≤ 5 minutes

Look for these stickers on instruments that have successfully met the validation criteria.

The instrument validation program is a collaborative effort with the major suppliers of multimode readers to insure that Transcreener assays perform optimally on your specific instrument. Scientists at BellBrook and/or the instrument vendor have tested Transcreener reagents with each instrument to determine optimal filters and settings for maximal assay performance. We provide you with application notes with all the important details, so you can be confident of success using Transcreener assays on the instrument of your choice.

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What's Included


Component	Notes
cGAMP Antibody-Terbium Conjugate	Sufficient antibody is included in the kit to complete 1,000 assays (Part #3025-1K) or 10,000 assays (Part #3025-10K). The final antibody concentration in the 20 µL reaction is 4 nM.
cGAMP ATTO 647 Tracer	The final tracer concentration in the 20 µL reaction is 8 nM.
Stop & Detect Buffer C, 10X	The Stop & Detect Buffer C components will stop enzyme reactions that require Mg²⁺. To ensure that the enzyme reaction is stopped completely, confirm that the EDTA concentration is at least equimolar to the magnesium ion concentration in the reaction. The final concentration of Stop & Detect Buffer C at the time of FRET measurement is 0.5X.
ATP	The ATP supplied in the kit can be used for the enzyme reaction and standard curve.
GTP	The GTP supplied in this kit can be used for the enzyme reaction and standard curve.
cGAMP	The cGAMP supplied in this kit can be used for a standard curve.
Interferon Stimulatory DNA	The double stranded interferon stimulatory DNA (ISD) is a 45-bp oligomer used to activate the cGAS enzyme.

What You Will Need


Component	Notes
cGAS Enzyme	Transcreener cGAMPassays are designed for use with purified enzyme preparations. Contaminating enzymes, such as phosphates and nucleotidases, can produce background signal and reduce the assay window.
Enzyme Buffer	User-supplied enzyme buffer components include enzyme, buffer, MgCl₂,Brij-35, and test compounds.
Assay Plates	Assay Plates Can Be Purchased Separately From BellBrook Labs. It is important to use assay plates that are entirely white with a nonbinding surface. We recommend Corning^® 384-well plates (Cat. #4513).
Plate Reader	A microplate reader configured to measure TR-FRET of the Tb:cGAMP ATTO 647 donor:acceptor pair is required.
Liquid Handling Devices	Use liquid handling devices that can accurately dispense a minimum volume of 2.5 µL into 384-well plates.
Ultrapure Water	Some deionized water systems are contaminated with nucleases that can degrade both nucleotide substrates and products, reducing assay performance. Careful handling and use of ultrapure water eliminates this potential problem.

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Transcreener cGAMP Assay Kits - TR-FRET Readout

* For custom or bulk orders (over 100,000 wells) please contact us (info@bellbrooklabs.com) for a quote.

Cat. No.	Product Description	Price	Add to Cart
3025-1K	Transcreener cGAMP TR-FRET Assay (1,000 Assay, 384 Well)	$919.00	Add to Cart
3025-10K	Transcreener cGAMP TR-FRET Assay (10,000 Assay, 384 Well)	$5,239.00	Add to Cart
3025-100K	Transcreener cGAMP TR-FRET Assay (10 X 10,000 Assay, 384 Well)	$26,195.00	Add to Cart

cGAS Screening Bundles - TR-FRET Readout

* Includes enough enzyme for respective wells when used at 1.75 μg/mL (30 nM)

Cat. No.	Product Description	Price	Add to Cart
3025-1K-B	Includes Transcreener cGAS TR-FRET Assay 1K Kit and 20 µg Human cGAS Enzyme	$1,419.00	Add to Cart
3025-10K-B	Includes Transcreener cGAS TR-FRET Assay 10K Kit and 200 µg Human cGAS Enzyme	$7,349.00	Add to Cart

Assay Plates for TR-FRET

Cat. No.	Product Description	Price	Add to Cart
2234	Corning 96-Well White Assay Plates (#3642), 3-Pack (200+ Assays)	$55.00	Add to Cart
2231	Corning 384-Well White Assay Plates (#4513), 3-Pack (1,000+ Assays)	$65.00	Add to Cart
2232	Corning 384-Well White Assay Plates (#4513), 30-Pack (10,000+ Assays)	$529.00	Add to Cart

cGAS Enzyme Ordering Information

* For custom or bulk orders (over 100 μg) please contact us (info@bellbrooklabs.com) for a quote.

Cat. No.	Product Description	Price	Add to Cart
2227	Recombinant cGAS Enzyme, Active, Human, Full-Length, 10 µg	$439.00	Add to Cart
2228	Recombinant cGAS Enzyme, Active, Human, Full-Length, 100 µg	$1,498.00	Add to Cart