Revvity, 64TIE2Y92PEH, HTRF Human Phospho-Tie2 (Tyr992) Detection Kit, 10,000 assay points

Overview
Phospho-Tie assay kits are designed for the robust quantification of Tie modulation, phosphorylated on Tyr992, upon angiopoietin activation. The simple mix-and-read protocol eliminates all wash steps for faster analysis and high quality output.
Tie2 is a tyrosine-protein kinase found in endothelial cells, serving as a cell-surface receptor for angiopoietins. It plays a crucial role in regulating angiogenesis, cell migration, survival, adhesion, and proliferation, primarily ensuring vascular stability and maintenance. When Angiopoietin 1 is present, Tie2 receptors form homodimers, leading to the autophosphorylation of the C-terminal tails at Tyrosine 992 and the activation of downstream signaling pathways.
How it works
Phospho-Tie2 (Tyr992) assay principle
The Phospho-Tie2 (Tyr992) assay measures Tie2 when phosphorylated at Tyr992. 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-Tie2 (Tyr992) 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-Tie2 (Tyr992) HTRF detection reagents. This protocol allows for the cells' viability and confluence to be monitored.

Phospho-Tie2 (Tyr992) one-plate assay protocol
Detection of phosphorylated Tie2 (Tyr992) 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-Tie2
Target Class-Phosphoproteins
Target Species-Human
Technology-TR-FRET
Therapeutic Area-Oncology
Unit Size-10,000 assay points