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HTRF Human RIG-I Binding Kit HTRF®

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

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

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

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Overview

A fast and easy way to identify new binders to human RIG-I. The Retinoic acid-Inducible Gene I (RIG-I) is a cytosolic pattern recognition receptor which is known to recognize RNA pathogens in cells. This protein is involved in the innate immune system and induces a type 1 interferon response. The RIG-I protein recognizes infected cells by detecting viral dsRNA. Once activated by a dsRNA, the N-terminus domain of RIG-I binds mitochondrial antiviral signaling proteins, thereby activating the IFN-1 signaling pathway.

Benefits

  • Study innate immunity
  • Identify anti-tumorigenic drugs

Assay principle

The HTRF RIG-I binding assay is a competitive assay format which uses a biotinylated 3 phosphate-dsRNA, a streptavidin-d2, a 6His tagged human RIG-I protein, and an anti 6His Cryptate-labeled antibody. The compound being tested competes with the biotinylated 3p-dsRNA, and thereby prevents FRET from occurring.

RIG-I assay principle

Assay protocol

The Human RIG-I 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 RIG-I protein is then added, followed by the dispensing of the HTRF reagents: The anti 6His antibody labeled with Terbium cryptate and the biotinylated-3p-dsRNA bound to d2-labeled streptavidin. The reagents labeled 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.

RIG-I assay protocol

Effect of 5' triphosphate vs blunt-end dsRNA for RIG-I binding

Two different ds-RNAs were tested in parallel in the HTRF RIG-I binding assay. The first ds-RNA displays a triphosphate in 5', the second does not. Serial dilutions of both RNAs were carried out in diluent, and then RIG-I protein was dispensed. HTRF detection reagents were then added to the well for the final detection step. Results were collected after 1 hour of incubation at room temperature. The affinity of the RIG-I protein was increased by the presence of the triphosphate in 5' of the dsRNA, as expected.

Effect of 5' triphosphate vs blunt-end dsRNA for RIG-I binding

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Plate Reader Requirement

Choosing the right microplate reader ensures you’ll get an optimal readout. Discover our high performance reader, or verify if your lab equipment is going to be compatible with this detection technology.

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