Revvity, 64FLT3TPEG, HTRF Human Total FLT3 Detection Kit, 500 Assay Points

Overview
The HTRF Human FLT3 (Fms-like tyrosine kinase 3) detection assay, also named FLK2, monitors Wild-Type FLT3 (FLT3 WT) and/or Internal Tandem Duplication (ITD) FLT3 mutant, and is used to detect the expression of endogenous or overexpressed FLT3 in various cells. FLT3 is a class III receptor tyrosine kinase expressed in immune cells and activated by its ligand, FLT3L. After FLT3L binding, the FLT3 receptor dimerizes and activates a variety of pathways, including the phosphatidylinositol 3-kinase (PI3K) and the MAPK pathways resulting in cell division, proliferation, and survival of immune cells. A dysregulation of the FLT3 pathways could lead to anarchic proliferation, immature immune cells, and oncogenic phenotypes. FLT3 is the most commonly mutated gene in Acute Myeloid Leukemia (AML). Mutations in the FLT3 gene lead to constitutive activation of the receptor kinase activity, or disrupt the autoinhibitory activity of the receptor. The ITD FLT3 mutant is well known for its implication in AML. FLT3 mutants have therefore become important drug targets for the pharmaceutical industry. The development of PROTAC compounds to target FLT3 degradation is a part of the therapeutic strategy to target forms of leukemia like AML.
How it works
HTRF Human Total FLT3 assay principle
The HTRF Human Total FLT3 assay quantifies the expression level of FLT3 in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Human Total FLT3 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 FLT3 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.

 

Human Total FLT3 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 Human Total FLT3 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

 

Human Total FLT3 one-plate assay protocol
Detection of Human FLT3 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
Brand-HTRF
Detection Modality-HTRF
Lysis Buffer Compatibility-Lysis Buffer 1 Lysis Buffer 2 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
Technology-TR-FRET
Unit Size-500 assay points