2′,3′-Cyclic GAMP STING-Based FRET Detection Kits

Research Areas

Documentation

Specifications

• Assay Type Detection Kit
• Sample Types Cell Lysates, Tissue Extracts, Tissue Culture Media
• Sensitivity 82.02 pmol/mL
• Species 2',3'-Cyclic GAMP is identical across species
• Samples/Plate 40 in duplicate
• Assay Duration 15 Minutes
• Readout Fluorescence, 458 nm Excitation and 600 nm/490 nm Emission
• Standard Curve
• Description

  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. The 2′,3′-Cyclic GAMP STING-Based FRET Detection Kit is a Detection Kit with a run time of 15 minutes. Please read the complete kit insert for more information before performing this assay.

  Use our provided 2’,3’-Cyclic GAMP Standard to generate a standard curve for the assay. Pipette the standards or diluted samples into a transparent microtiter plate. Add the 2’,3’-cGAMP BioSTING Protein to each well, tapping the plate to ensure sufficient mixing of reagents. Then, incubate the mixture at 37°C covered, shaking for 15 minutes. The fluorescent-generating reaction occurs between the BioSTING and the 2’,3’-cGAMP in the standard or sample. 

  After the 15-minute incubation, use a plate reader to detect and record the generated signal at 600 nm and 490. Use the intensity and the standard curve to calculate the 2’,3’-cGAMP concentration in the samples.

  Background:

  Pollock et al. 2020 used STING (stimulator of interferon genes) as the base to develop an in vivo/in vitro biosensor. Murine STING demonstrated a higher propensity to bind to bacterial and eukaryotic CDNs (cyclic dinucleotides) versus human STING, lending to potentially broader application. 

  This STING biosensor (BioSTING) is a fluorescence resonance energy transfer (FRET) based sensor designed to detect 2’,3’-cGAMP in real-time within living cells. FRET is a fluorescence detection platform based on the distance-dependent relationship between two fluorophores. In the case of BioSTING, mTFP, and mKO2 are the fluorophores of choice. When mTFP is excited with a wavelength of 458 nm, the emission is detected at 490 nm when no ligand (2’,3’-cGAMP) is present. When BioSTING binds to the ligand, the two fluorophores are closer, transferring the fluorescence from mTFP to mKO2 and changing the emission wavelength to 600 nm. Using this detection method, BioSTING shows applicability for in vitro high-throughput screening for CDN production modulation and direct screening for STING agonists and antagonists.