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

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

The human STING WT 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 WT (R232).

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.

Benefits

  • Study innate immunity
  • Discover STING agonists
  • Identify anti-tumorigenic drugs

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-principle-htrf-sting-human-binding-assay-64BDSTGPEG-64BDSTGPEH-64BDSTGPEJ

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.

assay-protocol-htrf-sting-human-binding-assay-64BDSTGPEG-64BDSTGPEH-64BDSTGPEJ
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.

Compound screening of various sting ligand using the HTRF STING binding assay

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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