Revvity, 64YESY4PEH, HTRF Human and Mouse Phospho-YES (Tyr426) Detection Kit, 10,000 Assay Points

Overview
YES, also known as YES1 which belongs to the Src family of kinases, is a proto-oncogene and a non-receptor tyrosine kinase located at the plasma membrane. Once activated, YES phosphorylates various downstream effectors including LATS1/2, YAP, FAK, Caveolin-1, and p130CAS, as well as Crk. The kinase contributes to a wide range of cellular processes, including cell proliferation, survival, cytoskeleton rearrangements, migration, and adhesion. Dysregulation of YES can contribute to pathological conditions, especially in the context of cancer.
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 time of readout or size of assays
How it works
Phospho-YES (Tyr426) assay principle
The phospho-YES (Tyr426) assay measures YES when phosphorylated at Tyr26. 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 leads to 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-YES (Tyr426) 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-YES (Tyr426) HTRF detection reagents. This protocol allows for the cells' viability and confluence to be monitored.

 

Phospho-YES (Tyr426) one-plate assay protocol
Detection of phosphorylated-YES(Tyr426) 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
Automation Compatible-Yes
Brand-HTRF
Detection Modality-HTRF
Lysis Buffer Compatibility-Lysis Buffer 4
Molecular Modification-Phosphorylation
Product Group-Kit
Sample Volume-16 µL
Shipping Conditions-Shipped in Dry Ice
Target-YES
Target Class-Phosphoproteins
Target Species-Human Mouse
Technology-TR-FRET
Therapeutic Area-Inflammation Oncology
Unit Size-10,000 assay points