Revvity, 64RETTPEH, HTRF Human and Mouse Total RET Detection Kit, 10,000 Assay Points

Overview
The Total-RET cellular assay monitors total RET (short and long isoforms), and can be used as a normalization assay with the Phospho-RET (pan) kit. This kit is compatible with the buffers from the Phospho-RET kit, so the same lysate can be used for analyses of both the phosphorylated and the total protein populations.
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
Total-RET assay principle
The HTRF Total-RET assay quantifies the expression level of RET in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Total-RET assay uses two labeled antibodies: one coupled to a donor fluorophore, the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In presence of RET 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-RET 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 Total-RET HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

 

Total-RET one-plate assay protocol
Detection of Total-RET 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-Total
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-10,000 assay points