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Met (Phospho-Tyr1349) Colorimetric Cell-Based ELISA Kit

SKU:
CBCAB01570
Product Type:
ELISA Kit
ELISA Type:
Cell Based Phospho Specific
Reactivity:
Human
Mouse
Rat
Detection Method:
Colorimetric
$719
Frequently bought together:

Description

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Met (Phospho-Tyr1349)Colorimetric Cell-Based ELISA Kit

The MET Phospho-Tyr1349 Colorimetric Cell-Based ELISA Kit is a cutting-edge tool for researchers seeking to accurately measure MET phosphorylation at tyrosine 1349 in cell lysates. This kit offers high sensitivity and specificity, allowing for precise and reproducible results in a variety of experimental settings.MET is a receptor tyrosine kinase that plays a key role in regulating cell growth, survival, and motility. Phosphorylation of MET at tyrosine 1349 is known to be involved in downstream signaling pathways that contribute to cancer progression and metastasis.

By measuring MET phosphorylation at this specific site, researchers can gain valuable insights into the molecular mechanisms underlying cancer development and potentially identify novel therapeutic targets.Overall, the MET Phospho-Tyr1349 Colorimetric Cell-Based ELISA Kit is an essential tool for studying MET signaling pathways and identifying potential therapeutic strategies for cancer treatment. Its high sensitivity and reliability make it a valuable asset for researchers in the field of cancer biology and drug development.

Product Name:Met (Phospho-Tyr1349) Colorimetric Cell-Based ELISA
Product Code:CBCAB01570
ELISA Type:Cell-Based
Target:Met (Phospho-Tyr1349)
Reactivity:Human, Mouse, Rat
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nm
Format:2 x 96-Well Microplates

The Met (Phospho-Tyr1349) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect Met protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated Met in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on Met phosphorylation.

Qualitative determination of Met (Phospho-Tyr1349) concentration is achieved by an indirect ELISA format. In essence, Met (Phospho-Tyr1349) is captured by Met (Phospho-Tyr1349)-specific primary (1ø) antibodies while the HRP-conjugated secondary (2ø) antibodies bind the Fc region of the 1ø antibody. Through this binding, the HRP enzyme conjugated to the 2ø antibody can catalyze a colorimetric reaction upon substrate addition. Due to the qualitative nature of the Cell-Based ELISA, multiple normalization methods are needed:

1. A monoclonal antibody specific for human GAPDH is included to serve as an internal positive control in normalizing the target absorbance values.
2. Following the colorimetric measurement of HRP activity via substrate addition, the Crystal Violet whole-cell staining method may be used to determine cell density. After staining, the results can be analysed by normalizing the absorbance values to cell amounts, by which the plating difference can be adjusted.
Database Information:Gene ID: 4233, UniProt ID: P08581, OMIM: 114550/164860/605074/611015, Unigene: Hs.132966
Gene Symbol:MET
Sub Type:Phospho
UniProt Protein Function:Receptor tyrosine kinase that transduces signals from the extracellular matrix into the cytoplasm by binding to hepatocyte growth factor/HGF ligand. Regulates many physiological processes including proliferation, scattering, morphogenesis and survival. Ligand binding at the cell surface induces autophosphorylation of MET on its intracellular domain that provides docking sites for downstream signaling molecules. Following activation by ligand, interacts with the PI3-kinase subunit PIK3R1, PLCG1, SRC, GRB2, STAT3 or the adapter GAB1. Recruitment of these downstream effectors by MET leads to the activation of several signaling cascades including the RAS-ERK, PI3 kinase-AKT, or PLCgamma-PKC. The RAS-ERK activation is associated with the morphogenetic effects while PI3K/AKT coordinates prosurvival effects. During embryonic development, MET signaling plays a role in gastrulation, development and migration of muscles and neuronal precursors, angiogenesis and kidney formation. In adults, participates in wound healing as well as organ regeneration and tissue remodeling. Promotes also differentiation and proliferation of hematopoietic cells. May regulate cortical bone osteogenesis.
NCBI Summary:This gene encodes a member of the receptor tyrosine kinase family of proteins and the product of the proto-oncogene MET. The encoded preproprotein is proteolytically processed to generate alpha and beta subunits that are linked via disulfide bonds to form the mature receptor. Further processing of the beta subunit results in the formation of the M10 peptide, which has been shown to reduce lung fibrosis. Binding of its ligand, hepatocyte growth factor, induces dimerization and activation of the receptor, which plays a role in cellular survival, embryogenesis, and cellular migration and invasion. Mutations in this gene are associated with papillary renal cell carcinoma, hepatocellular carcinoma, and various head and neck cancers. Amplification and overexpression of this gene are also associated with multiple human cancers. [provided by RefSeq, May 2016]
UniProt Code:P08581
NCBI GenInfo Identifier:251757497
NCBI Gene ID:4233
NCBI Accession:P08581.4
UniProt Secondary Accession:P08581,O60366, Q12875, Q9UDX7, Q9UPL8, A1L467, B5A932 E7EQ94,
UniProt Related Accession:P08581
Molecular Weight:85,745 Da
NCBI Full Name:Hepatocyte growth factor receptor
NCBI Synonym Full Names:MET proto-oncogene, receptor tyrosine kinase
NCBI Official Symbol:MET  
NCBI Official Synonym Symbols:HGFR; AUTS9; RCCP2; c-Met; DFNB97  
NCBI Protein Information:hepatocyte growth factor receptor
UniProt Protein Name:Hepatocyte growth factor receptor
UniProt Synonym Protein Names:HGF/SF receptor; Proto-oncogene c-Met; Scatter factor receptor; SF receptor; Tyrosine-protein kinase Met
Protein Family:C-methyltransferase
UniProt Gene Name:MET  
ComponentQuantity
96-Well Cell Culture Clear-Bottom Microplate2 plates
10X TBS24 mL
Quenching Buffer24 mL
Blocking Buffer50 mL
15X Wash Buffer50 mL
Primary Antibody Diluent12 mL
100x Anti-Phospho Target Antibody 60 µL
100x Anti-Target Antibody60 µL
Anti-GAPDH Antibody60 µL
HRP-Conjugated Anti-Rabbit IgG Antibody12 mL
HRP-Conjugated Anti-Mouse IgG Antibody12 mL
SDS Solution12 mL
Stop Solution24 mL
Ready-to-Use Substrate12 mL
Crystal Violet Solution12 mL
Adhesive Plate Seals2 seals

The following materials and/or equipment are NOT provided in this kit but are necessary to successfully conduct the experiment:

  • Microplate reader able to measure absorbance at 450 nm and/or 595 nm for Crystal Violet Cell Staining (Optional)
  • Micropipettes with capability of measuring volumes ranging from 1 µL to 1 ml
  • 37% formaldehyde (Sigma Cat# F-8775) or formaldehyde from other sources
  • Squirt bottle, manifold dispenser, multichannel pipette reservoir or automated microplate washer
  • Graph paper or computer software capable of generating or displaying logarithmic functions
  • Absorbent papers or vacuum aspirator
  • Test tubes or microfuge tubes capable of storing ≥1 ml
  • Poly-L-Lysine (Sigma Cat# P4832 for suspension cells)
  • Orbital shaker (optional)
  • Deionized or sterile water

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

StepProcedure
1.Seed 200 µL of 20,000 adherent cells in culture medium in each well of a 96-well plate. The plates included in the kit are sterile and treated for cell culture. For suspension cells and loosely attached cells, coat the plates with 100 µL of 10 µg/ml Poly-L-Lysine (not included) to each well of a 96-well plate for 30 minutes at 37 °C prior to adding cells.
2.Incubate the cells for overnight at 37 °C, 5% CO2.
3.Treat the cells as desired.
4.Remove the cell culture medium and rinse with 200 µL of 1x TBS, twice.
5.Fix the cells by incubating with 100 µL of Fixing Solution for 20 minutes at room temperature. The 4% formaldehyde is used for adherent cells and 8% formaldehyde is used for suspension cells and loosely attached cells.
6.Remove the Fixing Solution and wash the plate 3 times with 200 µL 1x Wash Buffer for five minutes each time with gentle shaking on the orbital shaker. The plate can be stored at 4 °C for a week.
7.Add 100 µL of Quenching Buffer and incubate for 20 minutes at room temperature.
8.Wash the plate 3 times with 1x Wash Buffer for 5 minutes each time.
9.Add 200 µL of Blocking Buffer and incubate for 1 hour at room temperature.
10. Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
11.Add 50 µL of 1x primary antibodies Anti-Met (Phospho-Tyr1349) Antibody, Anti-Met Antibody and/or Anti-GAPDH Antibody) to the corresponding wells, cover with Parafilm and incubate for 16 hours (overnight) at 4 °C. If the target expression is known to be high, incubate for 2 hours at room temperature.
12.Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
13.Add 50 µL of 1x secondary antibodies (HRP-Conjugated AntiRabbit IgG Antibody or HRP-Conjugated Anti-Mouse IgG Antibody) to corresponding wells and incubate for 1.5 hours at room temperature.
14.Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
15.Add 50 µL of Ready-to-Use Substrate to each well and incubate for 30 minutes at room temperature in the dark.
16.Add 50 µL of Stop Solution to each well and read OD at 450 nm immediately using the microplate reader.

(Additional Crystal Violet staining may be performed if desired – details of this may be found in the kit technical manual.)

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