Assay Principle: 

The 2′,3′-Cyclic GAMP STING-Based FRET Detection Kit quantitatively measures 2’,3’-cGAMP levels in cell lysates, tissue extracts, and tissue culture media. This detection kit has a run time of 15 minutes. Please read the complete kit insert for more information before performing this assay.

Protocol Summary:

• Transfer standards or diluted samples into the transparent microtiter plate provided in the kit.
• Add the 2’,3’-cGAMP BioSTING Protein to each well, ensuring thorough mixing.
• Incubate the mixture at 37°C, covered and shaking, for 15 minutes. The FRET-based reaction occurs between the BioSTING protein and the 2’,3’-cGAMP.
• After incubation, detect the fluorescent signal using a plate reader at 490 nm and 600 nm wavelengths. Calculate the 2’,3’-cGAMP concentration using the measured intensities and the standard curve.

Background:

Developed based on research by Pollock et al. in 2020, the BioSTING protein in this kit is a novel biosensor that uses fluorescence resonance energy transfer (FRET) for the detection of 2’,3’-cGAMP in real-time within living cells. FRET, a technique reliant on the proximity of two fluorophores, enables sensitive and dynamic measurement of molecular interactions. In BioSTING, the fluorophores mTFP and mKO2 work in tandem; the absence of 2’,3’-cGAMP results in an emission at 490 nm, while binding with the ligand brings the fluorophores closer, shifting the emission to 600 nm.

This STING biosensor demonstrates a higher affinity in murine models for binding both bacterial and eukaryotic cyclic dinucleotides (CDNs) compared to human variants, suggesting a broader range of applications. The DetectX® 2′,3′-Cyclic GAMP STING-Based FRET Detection Kit not only provides in vitro high-throughput screening for CDN modulation but also enables the direct screening of STING agonists and antagonists, making it a valuable tool in immunology and molecular biology research.