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Product Protocol Product SDS MyAssays Data Reduction Template

Specifications

  • Assay Type Competitive ELISA
  • Sample Types Cell Lysates, Tissue Extracts, Tissue Culture Media
  • Sensitivity 0.048 pmol/mL
  • Species 2',3'-Cyclic GAMP is identical across species
  • Assay Duration 2.5 Hours
  • Samples/Plate 183 in Duplicate
  • Readout Colorimetric, 450 nm
  • Standard Curve
  • Description

    Assay Principle:

    The 2′,3′-Cyclic GAMP ELISA Kit (384-Well Plate) quantitatively measures 2’,3’-cGAMP present in lysed cells and tissue, EDTA plasma, and tissue culture media samples. The 2′,3′-Cyclic GAMP ELISA Kit (384-Well Plate) is a competitive ELISA with a run time of 2.5 hours.  Please read the complete kit insert before performing this assay. 

    Use our provided 2’,3’-Cyclic GAMP (cGAMP) standard to generate a standard curve for the assay. Pipette the standards or diluted samples into a transparent microtiter plate coated with our goat anti-rabbit IgG antibody. Add the 2’,3’-cGAMP-peroxidase conjugate and the 2’,3’-cGAMP polyclonal rabbit antibody. The immunological reaction occurs between the anti-2’,3’-cGAMP polyclonal antibody, the 2’,3’-cGAMP antigen in the sample or standard, and the 2’,3’-cGAMP-peroxidase conjugate. As the cortisol concentration in the sample increases, the bound cortisol-peroxidase conjugate decreases, causing a decrease in signal and vice versa. We recommend a 2-hour incubation shaking at room temperature. 

    After the 2-hour incubation, wash away the excess 2’,3’-cGAMP-peroxidase and add the TMB substrate. The TMB substrate reacts with the bound cortisol-peroxidase conjugate generating a signal detected by a plate reader at 450nm. Use the intensity and the standard curve to calculate the cortisol concentration in the samples. 

    Background:

    2’,3’-Cyclic guanosine monophosphate–adenosine monophosphate (2’,3’-cGAMP)is a mammalian messenger molecule. 2’,3’-cGAMP was the first cyclic di-nucleotide found in metazoa. 2’,3’-cGAMP is a “noncanonical“ cGAMP due to the atypical 2’-5’ phosphodiester linkage between the guanosine and the adenosine. 2’,3’-Cyclic GAMP is a novel second messenger in innate immunity that regulates type I interferon (IFN) production. Produced in mammalian cells by cGAS (cGAMP synthase) in response to double-stranded DNA in the cytoplasm binding to cGAS, cGAMP binds to the stimulator of interferon genes (STING). Subsequently, STING induces the TBK1-IRF3-dependent production of IFN-β. This cGAS-cGAMP-STING pathway is critical in pathogen detection and physiological conditions such as metabolic dysregulation, autoimmunity, and cancer.