Revvity, 64RETYPEG, HTRF Human and Mouse Phospho-RET (Pan-Tyr) Detection Kit, 500 Assay Points

Overview
The HTRF Phospho-RET (pan) cell-based assay conveniently and accurately detects phosphorylated RET on Tyrosine residues. The assay measures both short and long RET isoforms.
The proto-oncogene RET (Rearranged during Transfection), also known as c-Ret, is a receptor tyrosine kinase primarily expressed as two isoforms of 1072 & 1114 amino acids. RET activation by Glial cell line-derived neurotrophic Family Ligands (GFL) leads to its dimerization and autophosphorylation on intracellular Tyrosine residues. RET is required for the development of the nervous system and several other tissues. RET alterations have been detected in numerous human cancers. The development of highly selective and potent RET kinase inhibitors is therefore of great interest to treat RET-altered cancers. Recently, RET has also been linked to neurodegeneration.
How it works
Phospho-RET (pan) assay principle
The Phospho-RET (pan) assay measures RET when phosphorylated on Tyrosines. 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 is selected for its specific binding to phosphorylated tyrosine residues, the second for its ability to recognize specifically 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-RET (pan) 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-RET (pan) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

 

Phospho-RET (pan) one-plate assay protocol
Detection of Phosphorylated RET (pan) 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
Automation Compatible-Yes
Brand-HTRF
Detection Modality-HTRF
Lysis Buffer Compatibility-Lysis Buffer 1 Lysis Buffer 2 Lysis Buffer 3 Lysis Buffer 4
Molecular Modification-Phosphorylation
Product Group-Kit
Sample Volume-16 µL
Shipping Conditions-Shipped in Dry Ice
Target Class-Phosphoproteins
Target Species-Human Mouse
Technology-TR-FRET
Therapeutic Area-Oncology & Inflammation
Unit Size-500 assay points