| Assay Method Information | |
| | WT STING Binding Assay |
| Description: | An assay format was optimized to demonstrate binding of recombinant 6×His-tagged human STING protein labeled with Terbium Cryptate by the natural ligand, 2′3′cGAMP labeled with d2 (the acceptor). Upon proximity of the two dyes, the excitation of the donor by the flash lamp on the PHERAstar FSX plate reader triggers a Fluorescence Resonance Energy Transfer (FRET) towards the acceptor, which in turn fluoresces at 665 nm. To assess the ability of the synthetic small molecule STING ligands to bind to human STING, a competitive assay format was applied. A 10-point titration of each of the synthetic ligands in 5 uL were transferred into a 384 well plate, followed by 20 uL of assay buffer containing the 6×His-tagged human STING protein and labeled 2′3′cGAMP ligand and incubated for three hours at room temperature. The raw values obtained from the PHERAstar were used to calculate the reported IC50 values (the signal is inversely proportional to the binding of the synthetic ligand) through curve fitting in Genedata. The percent inhibition was calculated based upon the maximal amount of binding by synthetic compound versus the maximum binding of unlabeled 2′3′ cGAMP which was used as a control in each assay. |
| Affinity data for this assay | |
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