Human Phospho-HGFR/c-MET DuoSet IC ELISA Summary
* Provided that the recommended microplates, buffers, diluents, substrates and solutions are used, and the assay is run as summarized in the Assay Procedure provided.
Product Features
- Optimized capture and detection antibody pairings and recommended concentrations save lengthy development time
- Development protocols are provided to guide further assay optimization
- Assay can be customized to your specific needs
- Available in 2, 5, and 15-(96-well) plate pack sizes
- Economical alternative to Western blot
Kit Content
- Capture Antibody
- Conjugated Detection Antibody
- Calibrated Immunoassay Standard or Control
Other Reagents Required
PBS: (Catalog # DY006), or 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2O4, pH 7.2 - 7.4, 0.2 µm filtered
Wash Buffer: (Catalog # WA126), or equivalent
Lysis Buffer*
IC Diluent*
Blocking Buffer*
Substrate Solution: 1:1 mixture of Color Reagent A (H2O2) and Color Reagent B (Tetramethylbenzidine) (Catalog # DY999)
Stop Solution: 2 N H2SO4 (Catalog # DY994)
Microplates: R&D Systems (Catalog # DY990), or equivalent
Plate Sealers: ELISA Plate Sealers (Catalog # DY992), or equivalent
*For the Lysis Buffer, IC Diluent, and Blocking BUffer recommended for a specific DuoSet ELISA Development Kit, please see the product
Scientific Data
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Figure 1. The Human Phospho-HGF R DuoSet IC ELISA is more sensitive than immunoprecipitation (IP)-Western analysis The human epidermoid carcinoma cell line, A431, was treated with 100 ng/mL recombinant human HGF for five minutes to induce tyrosine phosphorylation of HGF R. Serial dilutions of lysates were analyzed by (A) IP-Western blot and (B) this ELISA. IPs were done using an anti-HGF R polyclonal antibody and Protein G agarose. Immunoblots were incubated with a biotinylated anti-phosphotyrosine monoclonal antibody (Catalog # BAM1676) to detect phospho-HGF R (p-HGF R). Bands were visualized with Streptavidin-HRP (Cat # DY998) followed by chemiluminescent detection using WesternGloTM Chemiluminescent Detection Substrate (Catalog # AR004).
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Figure 2. The Human Phospho-HGF RDuoSet IC ELISA detects ligand-induced HGF R tyrosine phosphorylation A431 cells were untreated or treated with 100 ng/mL recombinant human HGF for five minutes. ELISA and IP-Western blot (inset) analyses were done using 50 μg and 500 μg of lysate, respectively. IP-Western blots for phospho-HGF R (p-HGF R) were done as described in Figure 1. Blots were stripped and total HGF R was detected using a biotinylated anti-HGF R polyclonal antibody (Catalog # BAF358).
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Figure 3. The specificity of the Human Phospho-HGF RDuoSet IC ELISA is confirmed by receptor competition A431 cells were treated with 100 ng/mL recombinant human HGF for five minutes. The indicated amounts of recombinant extracellular domains of human HGF R (Catalog #358-MT), human EGF R (Catalog #1095-ER) or human IGF-I sR (Catalog #305-GR) were added to 50 μg lysate and analyzed using this ELISA. Competition was observed only with recombinant HGF R.
Product Datasheets
Preparation and Storage
Background: HGFR/c-MET
HGF receptor, a product of the proto-oncogene c-met, is a heterodimeric transmembrane glycoprotein that is a receptor-type tyrosine kinase. c-MET is synthesized as a single-chain precursor (pr170) which undergoes post-translational glycosylation and proteolytic cleavage to give rise to the heterodimeric mature form.
Citations for Human Phospho-HGFR/c-MET DuoSet IC ELISA
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
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Citations: Showing 1 - 5
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Andrographolide enhanced 5-fluorouracil-induced antitumor effect in colorectal cancer via inhibition of c-MET pathway
Authors: M Su, B Qin, F Liu, Y Chen, R Zhang
Drug Des Devel Ther, 2017-11-23;11(0):3333-3341.
Species: Human
Sample Types: Cell Culture Supernates
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Shedding of c-Met ectodomain correlates with c-Met expression in non-small cell lung cancer.
Authors: Fu, Le, Guo, Wei, Liu, Bingshan, Sun, Linlin, Bi, Zhenghon, Zhu, Li, Wang, Xinyan, Liu, Bin, Xie, Qian, Li, Ke
Biomarkers, 2013-03-01;18(2):126-35.
Species: Human
Sample Types: Cell Culture Supernates
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Epidermal growth factor receptor regulates MET levels and invasiveness through hypoxia-inducible factor-1alpha in non-small cell lung cancer cells.
Authors: Xu L, Nilsson MB, Saintigny P, Cascone T, Herynk MH, Du Z, Nikolinakos PG, Yang Y, Prudkin L, Liu D, Lee JJ, Johnson FM, Wong KK, Girard L, Gazdar AF, Minna JD, Kurie JM, Wistuba II, Heymach JV
Oncogene, 2010-02-15;29(18):2616-27.
Species: Human
Sample Types: Cell Lysates
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Use of IHC and newly designed matriptase inhibitors to elucidate the role of matriptase in pancreatic ductal adenocarcinoma.
Authors: Uhland K, Siphos B, Arkona C, Schuster M, Petri B, Steinmetzer P, Mueller F, Schweinitz A, Steinmetzer T, Van De Locht A
Int. J. Oncol., 2009-08-01;35(2):347-57.
Species: Human
Sample Types: Cell Lysates
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Imbalance in the pro-hepatocyte growth factor activation system in bleomycin-induced lung fibrosis in mice.
Authors: Phin S, Marchand-Adam S, Fabre A, Marchal-Somme J, Bantsimba-Malanda C, Kataoka H, Soler P, Crestani B
Am. J. Respir. Cell Mol. Biol., 2009-05-15;42(3):286-93.
Species: Human
Sample Types: Cell Lysates
FAQs
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Which phosphorylated sites are recognized by this assay?
This assay utilizes an anti-phosphorylated tyrosine monoclonal detection antibody, and it recognizes all phosphorylated tyrosine residues.
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