The human STING binding assay is designed to select and characterize compounds that specifically bind human STING protein.

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  • No-wash
  • Low sample consumption
  • All inclusive kit

The human STING binding assay is designed to select and characterize compounds that specifically bind human STING protein.

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Overview

A fast and easy way to identify new binders to human STING.

Stimulator of interferon genes (STING), also known as transmembrane protein 173 (TMEM173), is a protein playing a major role in innate immunity. Upon intracellular cytosolic DNA release from pathogens such as viruses and bacteria, 2’-3’cGAMP binds to STING protein and triggers the secretion of type 1 interferon. Identifying new STING ligands is therefore a way to control immunity and fight autoinflammatory diseases.

Assay principle

The HTRF STING binding assay is a competitive assay format which uses d2-labeled STING ligand, a 6His tagged human STING protein, and an anti 6His Cryptate-labeled antibody. Your compound competes with the STING ligand-d2 and thereby prevents FRET from occurring.

Assay protocol

The Human STING binding assay can be run in a 96- or 384-well low volume white plate (20 µL final). As described here, samples or standards are dispensed directly into the assay plate, the human His-tagged STING protein is then added followed by the dispensing of the HTRF reagents: the anti 6His antibody labeled with Terbium cryptate and  the STING ligand labeled with d2. The reagents labelled with HTRF fluorophores may be pre-mixed and added in a single dispensing step. No washing steps are needed. The protocol can be further miniaturized or upscaled by simply resizing each addition volume proportionally.

STING ligand-d2 - Kd15 nM
STING ligand-d2 - Concentration15 nM
2’3’ cGAMP Standard - IC505 nM
2’3’ cGAMP Standard - Ki2.5 nM
Signal to Background12.4

Compound screening

Various compounds known as STING ligands were added to the assay. 2’-3’ cGAMP (assay standard), cyclic di-AMP, and cyclic di-GMP display the right potency in good correlation with the literature. DMXAA, a non-specific compound, also well-known mouse STING binder, has no effect on the assay, confirming the human specificity of the kit.

STING simplified pathway

STING, for STimulator of INterferon Genes, is a cytoplasmic homodimeric protein localized in the endoplasmic reticulum which plays an essential role in innate immunity. Upon pathogen infection or mitochrondrial shrinking during apoptosis, floating dsDNAs bind and activate a DNA sensor, the cyclic GMP-AMP synthase (cGAS). Activated cGAS leads to the production of 2’-3’cGAMP, a cyclic dinucleotide, which then binds to STING proteins. In turn, phosphorylated STING interacts with TANK-binding-kinase-I (TBK1), leading to the recruitment and activation of the active interferon regulatory factor (IRF3) dimer. Nuclear translocation of the IRF3 dimer leads to the transcription of genes encoding IFN-α/β. In addition, the STING pathway controls NF-κB dependent inflammatory cytokine expression. As a negative feedback loop, the DNA-stimulated cGAS-STING-TBK1 pathway also triggers STING protein degradation through p62 SQSTM1 associated autophagy to switch off IFNb production.

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