Revvity, 64BDKEAP1PEG, HTRF Human KEAP1 Binding Kit, 500 Assay Points

Overview
The KEAP1-NRF2-ARE pathway plays an important role in the response to oxidative stress and in maintaining redox homeostasis. KEAP1 (Kelch-like ECH-associated protein 1) is an adaptor subunit of the Cullin 3-based E3 ubiquitin ligase, involved in oxidative stress response. By promoting UPS mediated degradation, KEAP1 controls the expression level of NRF2, a key transcription that regulates the expression of antioxidant proteins, therefore ensuring cells' protection from oxidative stress. Sustained activation of NRF2 has been associated with cancer resistance, and its inhibition has been shown to weaken drug resistance. Targeted protein degradation (TPD) uses small molecules to recruit E3 ubiquitin ligases into the proximity of the targeted protein of interest, promoting its ubiquitination-dependent degradation. Exploiting KEAP1 inhibitors for the development of bifunctional Proteolysis-targeting chimeras (PROTACs) is expected to expand the toolbox of E3 ligases to the removal of undesired proteins (e.g. NRF2) involved in various diseases such as cancers, neurodegenerative diseases, and metabolic disorders.
How it works
Assay principle
The HTRF Human KEAP1 Binding Kit is a competitive assay format implementing a KEAP1-Red Ligand, a GST-tagged human KEAP1 protein complex, and an anti GST Terbium Cryptate-labeled antibody. KEAP1 binding compounds compete with the KEAP1-Red Ligand and thereby prevent FRET from occurring.

 

Assay protocol
The KEAP1 binding assay can be run in 96- or 384-well low volume white plates (20 µL final). As described here, samples or standards are dispensed directly into the assay plate. The GST-tagged KEAP1 protein complex is then added, followed by the dispensing of the HTRF reagents: The anti GST antibody labeled with Terbium cryptate and the KEAP1 Ligand labeled with a Red HTRF acceptor. 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. Application-Protein Quantification
Brand-HTRF
Detection Modality-HTRF
Product Group-Kit
Sample Volume-5 µL
Shipping Conditions-Shipped in Dry Ice
Target Class-Biomarkers
Target Species-Human
Technology-TR-FRET
Unit Size-500 assay points