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

Transcreener cGAMP Assay (FP) Method

Ordering Information

Instrument Compatibility

Related Resources

Transcreener cGAMP Assay Kit - FP Readout

The Transcreener® cGAMP Assay with FP readout measures cGAMP via antibodies selective to cGAMP and a far-red fluorescent tracer. cGAMP produced in the reaction competes with the tracer, decreasing fluorescent properties and providing a fluorescent readout. The universal detection method allows screening of virtually any cGAMP-producing enzyme in a high throughput format.

The assay shows excellent data quality (Z' ≥ 0.7) and signal (≥85 mP polarization shift) at cGAMP ranges from 0.5 μM to 100 μM. Both reagents & signal are stable at room temperature for over 24 hours, providing outstanding flexibility for automated high-volume screening programs.

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

Fluorescence Polarization

Learn More About the Transcreener cGAMP Assay

Transcreener cGAMP FP 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

Achieve Robust Results

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

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

Important Messenger in the STING Pathway

DNA in the cytosol of eukaryotic cells indicates infection of 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.

Screen for Novel cGAS Modulators

Pilot screen from Life Chemicals Diversity Library. 1600 compounds screened with 10 nM cGAS enzyme and 100 µM ATP, 100 µM GTP, and 62.5 nm interferon stimulatory DNA. The enzyme reaction was 1 hour and plates were read on a BMG Pherastar® Plus plate reader.

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Accurate Dose Response Results

DRC using known inhibitor, G150. Here, we used 100 μM ATP, 100 μM GTP, 100 nM ISDNA, 20 mM Tris (pH 7.5), 5 mM MgCl₂, & 0.01% Brij-35 (pH 7.5). The Detection Mix included 1X Stop & Detect Buffer B, 8 nM cGAMP 633 Tracer, & 2.0 μg/mL cGAMP antibody.

cGAS Assay DRC with G150 inhibitor in FP Readout

IC₅₀ [G150] = 0.04 μM

Far Red FP Readouts Validated on Major Multimode Readers

Supplier	Instrument	FP Assays
	TriStar²S LB 942	In Review
	Tristar 5	In Review
	Mithras² LB 943	In Review

	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	Not Capable
	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	Not Capable
	SpectraMax^® Paradigm	Validated
	SpectraMax^® iD3/iD5	Validated
	SpectraMax^® i3x	Validated

	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
Δ mP ≥ 95 mP at 10% conversion of 10 μM ATP
Z' and Δ mP specifications to be met using Transcreener ADP² Assay Reagents
Read time to achieve Z' and Δ mP 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	Sufficient antibody is included in the kit to complete 1,000 assays (Cat. #3024-1K) or 10,000 assays (Cat. #3024-10K).
cGAMP ATTO 633 Tracer	The final tracer concentration in the 20 µL reaction is 4 nM.
Stop & Detect Buffer B, 10X	The Stop & Detect Buffer B 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 B at the time of FP 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 a 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^® cGAMP assays are designed for use with purified enzyme preparations. Contaminating enzymes, such as phosphatases or nucleotidases, can produce background signal and reduce the assay window
Enzyme Buffer	User-supplied enzyme buffer components include enzyme, buffer, MgCl_2,Brij-35, and test compounds.
Assay Plates	Assay Plates Can Be Purchased Separately From BellBrook Labs. FP - An entirely black plate with a non-binding surface is required. We suggest Corning 384-well plates Cat. #4514.
Plate Reader	A multidetection microplate reader configured to measure FP of the cGAMP ATTO 633 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 - FP 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
3024-1K	Transcreener cGAMP FP Assay (1,000 Assay, 384 Well)	$839.00	Add to Cart
3024-10K	Transcreener cGAMP FP Assay (10,000 Assay, 384 Well)	$4,849.00	Add to Cart
3024-100K	Transcreener cGAMP FP Assay (10 X 10,000 Assay, 384 Well)	$24,245.00	Add to Cart

cGAS Screening Bundles - FP Readout

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

Cat. No.	Product Description	Price	Add to Cart
3024-1K-B	Includes Transcreener cGAS FP Assay 1K Kit and 20 µg Human cGAS Enzyme	$1,339.00	Add to Cart
3024-10K-B	Includes Transcreener cGAS FP Assay 10K Kit and 200 µg Human cGAS Enzyme	$6,999.00	Add to Cart

Assay Plates for FP

Cat. No.	Product Description	Price	Add to Cart
2233	Corning 96-Well Black Assay Plates (#3686), 3-Pack (200+ Assays)	$55.00	Add to Cart
2229	Corning 384-Well Black Assay Plates (#4514), 3-Pack (1,000+ Assays)	$60.00	Add to Cart
2230	Corning 384-Well Black Assay Plates (#4514), 30-Pack (10,000+ Assays)	$479.00	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