Revvity, 64FLT3Y1PEH, HTRF Human Phospho-FLT3 (Tyr589/591) Detection Kit, 10,000 Assay Points

Overview
The HTRF Phospho Tyr 589/591 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 phosphorylation at Tyr 589/591 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.
How it works
Phospho-FLT3 (Tyr 589/591) assay principle
The Phospho-FLT3 (Tyr 589/591) assay measures FLT3 when phosphorylated at Tyrosines 589 and 591. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer.
The Phospho-FLT3 (Tyr 589/591) 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 the two 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-FLT3 (Tyr 589/591) two-plate assay protocol
The two-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates to a 384-well low volume detection plate before the addition of the Phospho-FLT3 Tyr589/591 HTRF detection reagents.
This protocol enables the cells' viability and confluence to be monitored.

 

Phospho-FLT3 (Tyr 589/591) one-plate assay protocol
Detection of Phospho-FLT3 Tyr589/591 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 4
Molecular Modification-Phosphorylation
Product Group-Kit
Sample Volume-16 µL
Shipping Conditions-Shipped in Dry Ice
Target Class-Phosphoproteins
Target Species-Human
Technology-TR-FRET
Unit Size-10,000 assay points