Revvity, 64AT3V2PEG, HTRF High Performance Human and Mouse phospho-STAT3 Tyr705 Detection Kit, 500 assay points

Overview
This High Performance HTRF cell-based assay enables rapid, quantitative, and highly sensitive detection of STAT3 phosphorylated on Tyr705 as a readout of JAK/STAT signaling activity. Compared to the standard version, the High-Performance format delivers a higher signal-to-background ratio and improved sensitivity, ensuring more robust results even in challenging conditions. These enhancements make it possible to obtain reliable results on more physiological models such as primary cells, broadening the applicability of the assay for translational and advanced research. STAT3 is upregulated in various diseases, particularly autoimmune disorders, neurodegenerative diseases, or cancers, making this kit a powerful tool for therapeutic strategies by inhibiting the pathway.
HTRF assays offer many advantages over other technologies:

Homogeneous add-and-read format
No wash steps
Low background
Straightforward miniaturization from 96- or 384-well microplates to high density assay formats such as 384-well low volume and 1536-well plates
Stable signal, providing flexibility in readout time or size of assays
How it works
High Performance Phospho-STAT3 (Tyr705) assay principle
The High Performance phospho-STAT3 (Tyr705) assay measures STAT3 when phosphorylated at Tyr705. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. It uses two antibodies: one labeled with a donor fluorophore and the other with an acceptor. The first antibody specifically binds the phosphorylated motif on the protein, while the second recognizes the protein independently of its phosphorylation state. Protein phosphorylation enables the formation of an immune complex involving both labeled antibodies, bringing the donor fluorophore into close proximity to the acceptor and generating a FRET signal. Its intensity is directly proportional to the concentration of phosphorylated protein in the sample, allowing assessment of the protein’s phosphorylation state in a no-wash assay format.

High Performance Phospho-STAT3 (Tyr705) 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 High Performance phospho-STAT3 (Tyr705) HTRF detection reagents. This protocol allows the cells’ viability and confluence to be monitored.

High Performance Phospho-STAT3 (Tyr705) one-plate assay protocol
Detection of phosphorylated STAT3 (Tyr705) 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 Lysis Buffer 3 Lysis Buffer 4
Molecular Modification-Phosphorylation
Product Group-Kit
Sample Volume-16 µL
Shipping Conditions-Shipped in Dry Ice
Target-STAT3
Target Class-Phosphoproteins
Target Species-Human Mouse
Technology-TR-FRET
Unit Size-500 assay points