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.

Specificity of Phospho-FLT3 (Tyr 589/591) assay

FLT3 Stable Ba/F3 cells were plated at 250,000 cells per well in a 96-well culture-treated plate in complete culture medium, and incubated overnight at 37°C, 5% CO2. Cells were treated with a high concentration of FLT3 ligand (FLT3-L) for 15 min at 37 °C 5% CO2, in the presence or absence of 1µM phosphorylated peptide surrounding the phosphorylation sites tyr 589 and tyr 591. They were then lysed with 50 µl of supplemented lysis buffer #4 (1X) for 30 min at RT under gentle shaking.

After cell lysis, 16 µL of lysate were transferred into a 384-well low volume white microplate, and 4 µL of the HTRF Phospho FLT3 Tyr 589/591 detection reagents were added. The HTRF signal was recorded after 3h of incubation at room temperature.

As expected, the phosphorylated peptide competes with the phosphorylated site of the FLT3 receptor, thus triggering a complete loss of signal. This demonstrates the specificity of the assay.

HTRF Phospho-FLT3 (Tyr 589/591) assay compared to Western Blot

FLT3 Stable Ba/F3 cells were cultured in a T175 flask in complete medium at 37°C, 5% CO2 to confluency. The cells were treated with 0.5 µg/mL of FLT3 ligand for 15 min at 37°C, 5% CO2.

AAfter medium removal, the cells were lysed with 3 mL of supplemented lysis buffer #4 (1x) for 30 min at RT under gentle shaking.

Serial dilutions of the cell lysate were performed using supplemented lysis buffer #4 (1x), and then 16µL of pure sample and of each dilution were transferred into a 384-well small volume microplate before the addition of 4µL of HTRF Phospho FLT3 Tyr 589/591 detection reagents. Signals were recorded after overnight incubation. /p>

Equal amounts of lysates were loaded into a gel for a side-by-side comparison between HTRF and Western Blot.

In these conditions, the HTRF Phospho FLT3 Tyr 589/591 assay is as least 16 times more sensitive than Western Blot.

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 the survival of immune cells. A dysregulation of the FLT3 pathways could lead to anarchic proliferation, immature immune cells, and oncogenic phenotypes. Mutations in the FLT3 gene lead to constitutive activation of the receptor kinase activity or to a disruption of the autoinhibitory activity of the receptor. The ITD FLT3 mutant does not require FLT3L to activate its downstream pathways. FLT3 is well known for its implication in the development of leukemia, and more specifically in Acute Myeloid Leukemia (AML).