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HTRF Human Phospho-MET Tyr1234/1235 Detection Kit

HTRF Human Phospho-MET Tyr1234/1235 Detection Kit HTRF®

The HTRF Human Phospho-MET Tyr1234/1235 detection kit is designed to monitor the expression level of cellular MET phosphorylated at Tyrosine 1234/1235.

All inclusive kit
Low sample consumption
No-wash
High sensitivity

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The HTRF Human Phospho-MET Tyr1234/1235 detection kit is designed to monitor the expression level of cellular MET phosphorylated at Tyrosine 1234/1235.

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Overview

c-MET, also called tyrosine-protein kinase MET or hepatocyte growth factor receptor (HGFR), is a human MET gene-coded protein with tyrosine kinase activity. It acts as a tyrosine kinase receptor, with functions related to wound healing, organogenesis, and embryonic development.

MET is expressed in epithelial cells and is triggered by hepatocyte factors HGF and HSF, with ramifications into various pathways like RAS-MAPK, AKT, and STAT3 signaling.

Abnormal activation of MET is linked to cancer progression, with effects on tumor growth, angiogenesis, and tumor cell migration & invasion. These relationships between MET and tumor cells are found across multiple cancer types (liver, brain, breast, kidney, etc) and are suspected to originate from a hijacking of MET and its ligand expression regulation, which leads to their overexpression and autocrine activation of MET.

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HTRF Phospho MET Tyr1234/1235 assay principle

The HTRF Phospho MET Tyr1234/1235 assay measures MET when phosphorylated on the Tyrosine 1234/1235. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer.

The assay uses 2 labeled antibodies: one with a donor fluorophore, 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 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.

Principle of the HTRF (h) Phospho-Y1234/1235 MET assay

HTRF Phospho MET Tyr1234/1235 2-plate assay protocol

The 2 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 the Phospho MET Tyr1234/1235 HTRF detection reagents.

This protocol enables the cells' viability and confluence to be monitored.

2-plate protocol of the HTRF Human Phospho-Y1234/1235 MET assay

HTRF Phospho MET Tyr1234/1235 one-plate assay protocol

Detection of phospho MET Tyr1234/1235 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.

1-plate protocol of the HTRF Human Phospho-Y1234/1235 MET assay

Validation of Phospho & Total MET detection in HGF-stimulated A431 cells

A431 cells were plated under 10µL in a 96-well plate (100,000 cells/well) in complete culture medium, and incubated overnight at 37ºC, 5% CO2. The next day, medium was removed and 50µL of complete medium was added before a 2h incubation at 37ºC, 5% CO2. Following the incubation, the cells were treated with 50µL of increasing concentrations of human HGF diluted in complete medium for 15 minutes at 37ºC, 5% CO2.

The medium was removed, and cells were then lysed with 60 µL of lysis buffer #2 and phospho-total protein blocking reagent for 45 minutes at RT under gentle shaking. After lysis, 16µL of lysate were transferred into a low volume white microplate before the addition of 2µL each of the donor and acceptor HTRF phospho-MET (Y1234/1235) or total MET detection reagents. The HTRF signal was recorded after an overnight incubation.

Validation of Phospho & Total MET detection in HGF-stimulated A431 cells

MET simplified pathway

When the tyrosine kinases of the c-MET docking sites becomes phosphorylated and active following HGF or HSF binding, they recruit a plethora of effectors (GRB2, SHC, CRK, CRKL, P13K, PLC-gamma, SRC, and STAT3). All these effectors signal in their respective directions and promote several pathways, making c-MET a multifunctional signaling hub. Such pathways and their effects include proliferation and cell-cycle progression through the RAS-MAPK pathway, the AKT survival pathway, dimerization and translocation of STAT3 to the nucleus with invasion effects, and a PLC-gamma-driven regulation loop for c-MET.

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How to run a cell based phospho HTRF assay

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In collaboration with Bayer - Scientific Presentations

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3' video to set up your Phospho assay - Videos

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Seeding and lysing recommendations for a number of cell culture vessels. - Technical Notes

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Learn how to reduce time and sample consumption - Application Notes

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Product Insert MET p-Y1234/1235 Kit / 64METY34PEG-64METY34PEH

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Safety Data Sheet (DEU) MET p-Y1234/1235 Kit / 64METY34PEG