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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.

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  • All inclusive kit All inclusive kit
  • Low sample consumption Low sample consumption
  • No-wash No-wash
  • High sensitivity High sensitivity

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.

Benefits

  • SPECIFICITY
  • PRECISION

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

HTRF cellular phospho-protein assays

Physiologically relevant results fo fast flowing research - Flyers

Best practices for analyzing brain samples with HTRF® phospho assays for neurosciences

Insider Tips for successful sample treatment - Technical Notes

Optimize your HTRF cell signaling assays on tissues

HTRF and WB compatible guidelines - Technical Notes

Best practices for analyzing tumor xenografts with HTRF phospho assays

Protocol for tumor xenograft analysis with HTRF - Technical Notes

Key guidelines to successful cell signaling experiments

Mastering the art of cell signaling assays optimization - Guides

HTRF® cell signaling platform combined with iCell® Hepatocytes

A solution for phospho-protein analysis in metabolic disorders - Posters

HTRF phospho-assays reveal subtle drug-induced effects

Detailed protocol and direct comparison with WB - Posters

Universal HTRF® phospho-protein platform: from 2D, 3D, primary cells to patient derived tumor cells

Analysis of a large panel of diverse biological samples and cellular models - Posters

HTRF phospho assays reveal subtle drug induced effects in tumor-xenografts

Tumor xenograft analysis: HTRF versus Western blot - Application Notes

HTRF cell-based phospho-protein data normalization

Valuable guidelines for efficiently analyzing and interpreting results - Application Notes

HTRF phospho-total lysis buffer: a universal alternative to RIPA lysis buffers

Increased flexibility of phospho-assays - Application Notes

HTRF Alpha-tubulin Housekeeping kit

Properly interpret your compound effect - Application Notes

Simplified pathway dissection with HTRF phospho-assays and CyBi-felix liquid handling

Analyse of PI3K/AKT/mTor translational control pathway - Application Notes

How to run a cell based phospho HTRF assay

What to expect at the bench - Videos

Unleash the potential of your phosphorylation research with HTRF

A fun video introducing you to phosphorylation assays with HTRF - Videos

How to run a cell based phospho HTRF assay

3' video to set up your Phospho assay - Videos

Guidelines for Cell Culture and Lysis in Different Formats Prior to HTRF Detection

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

Methodological Aspects of Homogeneous Time-Resolved Fluorescence (HTRF)

Learn how to reduce time and sample consumption - Application Notes

Assessment of drug efficacy and toxicity by combining innovative technologies

Combination of AlphaLISA®, HTRF®, or AlphaLISA® SureFire® Ultra™ immunoassays with the ATPlite™ 1step cell viability assay - Application Notes

Manual MET p-Y1234/1235 Kit / 64METY34PEG-64METY34PEH

64METY34PEG-64METY34PEH - Product Insert

Plate Reader Requirement

Choosing the right microplate reader ensures you’ll get an optimal readout. Discover our high performance reader, or verify if your lab equipment is going to be compatible with this detection technology.

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