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

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

cGAMP Assay Schematic - FP Readout v2

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

Results from cGAS Pilot Screen

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. 

Mix-and-Read-GAP-Assay

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.

cGAS STING Pathway

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.

cGAMP Assay Pilot Screen

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

SupplierInstrumentFP Assays
TriStar2S LB 942In Review
Tristar 5In Review
Mithras2 LB 943In Review
CytationTM 5Validated
CytationTM 3Validated
CytationTM 1Validated
SynergyTM H1Validated
SynergyTM 2/H4/4Click Me ArrowValidated
SynergyTM HTXNot Capable
SynergyTM Neo 2Validated
POLARstar® OmegaClick Me ArrowValidated
FLUOstar® OmegaNot Capable
PHERAstar® FSXClick Me ArrowValidated
PHERAstar® Plus/FSClick Me ArrowValidated
CLARIOstar® /PlusClick Me ArrowValidated
VANTAstarTMClick Me ArrowValidated
SenseIn Review
Analyst® GT/HTClick Me ArrowValidated
Gemini® XPS/EMNot Capable
SpectraMax® M2/M2eNot Capable
SpectraMax® M5/M5e/FlexStation® 3Not Capable
SpectraMax® ParadigmClick Me ArrowValidated
SpectraMax® iD3/iD5Click Me ArrowValidated
SpectraMax® i3xClick Me ArrowValidated
EnVision®/EnVision® XciteClick Me ArrowValidated
Infinite® M1000/M1000Pro/Safire2TMClick Me ArrowValidated
Infinite® M200Not Capable
Infinite® F500Click Me ArrowValidated
Infinite® F200/UltraevolutionClick Me ArrowContact Us
SparkTM 10MClick Me ArrowValidated

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. 

What's Included

What You Will Need

Transcreener cGAMP Assay Kits - FP Readout

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

cGAS Screening Bundles - FP Readout

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

Assay Plates for FP

cGAS Enzyme Ordering Information

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

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