Revvity, 64XBPTPEG, HTRF Human Total XBP1 Detection Kit, 500 Assay Points

Overview
The HTRF Human XBP1 detection assay monitors spliced XBP1 (XBP1s) as well as unspliced XBP1 (XBP1u), and is used to detect the expression of endogenous or overexpressed global XBP1 (XBP1s + XBP1u) in various cells.
Upon ER stress and accumulation of unfolded proteins, BiP releases its inhibitory binding to activate IRE1. IRE1 receptor dimerizes then autophosphorylates which activates its mRNase activity. Through this action, IRE1 splices XBP-1u mRNA to XBP1s, enabling its translation and then its translocation to the nucleus. XBP-1s then acts as a transcription factor.
How it works
Total XBP1 assay principle
The HTRF Human Total XBP1 assay quantifies the expression level of XBP1 (XBP1u + XBP1s) in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Human Total XBP1 assay uses two labeled antibodies, one coupled to a donor fluorophore and the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In presence of XBP1s or XBP1u in a cell extract, the addition of these conjugates brings the donor fluorophore into close proximity with the acceptor, and thereby generates a FRET signal. Its intensity is directly proportional to the concentration of the protein present in the sample, and provides a means of assessing the protein’s expression under a no-wash assay format.

Total XBP1 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 Human Total XBP1 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Total XBP1 one-plate assay protocol
Detection of Human Total XBP1 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 enables miniaturization while maintaining robust HTRF quality. Application-Cell Signaling
Brand-HTRF
Detection Modality-HTRF
Lysis Buffer Compatibility-Lysis Buffer 1 Lysis Buffer 2 Lysis Buffer 4
Molecular Modification-Total
Product Group-Kit
Sample Volume-16 µL
Shipping Conditions-Shipped in Dry Ice
Target Class-Phosphoproteins
Target Species-Human
Technology-TR-FRET
Unit Size-500 assay points