Revvity, 64IRE1S24PEH, HTRF Human Phospho-IRE1 (Ser724) Detection Kit, 10,000 assay points

Overview
IRE1 (Inositol requiring enzyme 1 alpha) is a serine/threonine kinase acting as one of three branches of the Unfolded Protein Response (UPR) signaling pathway with roles in regulation of ER stress and UPR. IRE1 is encoded by the ERN1 gene and is ubiquitously expressed in mammals. IRE1 is a type I transmembrane protein, exhibiting an ER luminal domain that senses the protein folding status and a catalytic kinase and RNase cytosolic domain. In recent years, IRE1 has emerged as a relevant therapeutic target in various diseases including degenerative disorders, inflammatory and metabolic pathologies, and cancer.
How it works
Phospho-IRE1 (Ser724) assay principle
The Phospho-IRE1 (Ser724) assay measures DDR1 when phosphorylated at Ser724. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The assay uses 2 antibodies, one labeled with a donor fluorophore and the other with an acceptor. The first antibody was selected for its specific binding to the phosphorylated motif on the protein, and the second for its ability to recognize the protein independently of its phosphorylation state. Protein phosphorylation enables an immune-complex formation involving both labeled antibodies, and which brings the donor fluorophore into close proximity to the acceptor, thereby generating a FRET signal.
Its intensity is directly proportional to the concentration of phosphorylated protein present in the sample and provides a means of assessing the protein's phosphorylation state under a no-wash assay format.

Phospho-IRE1 (Ser724) two-plate assay protocol
The two-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates into a 384-well low volume detection plate before the addition of Phospho-IRE1 (Ser724) HTRF detection reagents. This protocol allows for the cells' viability and confluence to be monitored.

Phospho-IRE1 (Ser724) one-plate assay protocol
Detection of Phosphorylated IRE1 (Ser724) with HTRF reagents can be performed in a single plate used for culturing, stimulation, and lysis. No washing steps are required. This HTS designed protocol allows miniaturization while maintaining robust HTRF quality. Application-Cell Signaling
Brand-HTRF
Detection Modality-HTRF
Lysis Buffer Compatibility-Lysis Buffer 1
Molecular Modification-Phosphorylation
Product Group-Kit
Sample Volume-16 µL
Shipping Conditions-Shipped in Dry Ice
Target-IRE1
Target Class-Phosphoproteins
Target Species-Human
Technology-TR-FRET
Therapeutic Area-Central Nervous System Inflammation Oncology
Unit Size-10,000 assay points