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HTRF Human Phospho-MET (Thr1234/1235) Detection Kit, 500 Assay Points
HTRF Human Phospho-MET (Thr1234/1235) Detection Kit, 500 Assay Points
generic HTRF phospho primary image
The HTRF Human Phospho-MET Tyr1234/1235 detection kit is designed to monitor the expression level of cellular MET phosphorylated at Tyrosine 1234/1235.
| Feature | Specification |
|---|---|
| Application | 細胞シグナル伝達 |
| Sample Volume | 16 µL |
The HTRF Human Phospho-MET Tyr1234/1235 detection kit is designed to monitor the expression level of cellular MET phosphorylated at Tyrosine 1234/1235.
Product variants
Unit Size: 500 assay points
Part #:
64METY34PEG
Unit Size: 10,000 assay points
Part #:
64METY34PEH
For research use only. Not for use in diagnostic procedures. All products to be used in accordance with applicable laws and regulations including without limitation, consumption, and disposal requirements under European REACH regulations (EC 1907/2006).
HTRF Human Phospho-MET (Thr1234/1235) Detection Kit, 500 Assay Points
generic HTRF phospho primary image
HTRF Human Phospho-MET (Thr1234/1235) Detection Kit, 500 Assay Points
Product information
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.
How it works
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.
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.
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.
Assay validation
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.
Specifications
| Application |
Cell Signaling
|
|---|---|
| Brand |
HTRF
|
| Detection Modality |
HTRF
|
| Lysis Buffer Compatibility |
Lysis Buffer 2
|
| 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 |
500 assay points
|
Video gallery
HTRF Human Phospho-MET (Thr1234/1235) Detection Kit, 500 Assay Points
HTRF Human Phospho-MET (Thr1234/1235) Detection Kit, 500 Assay Points
Resources
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Brochure
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