Human Phospho-HGFR/c-MET DuoSet IC ELISA
R&D Systems, part of Bio-Techne | Catalog # DYC2480-2
Key Product Details
Assay Type
Solid Phase Sandwich ELISA
Sample Type
Cell Lysates
Reactivity
Human
Human Phospho-HGFR/c-MET DuoSet IC ELISA 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
Product Summary for Human Phospho-HGFR/c-MET DuoSet IC ELISA
This DuoSet IC ELISA contains the basic components required for the development of sandwich ELISAs to measure tyrosine-phosphorylated human HGF R / c-MET in cell lysates. An immobilized capture antibody specific for HGF R / c-MET binds both phosphorylated and unphosphorylated HGF R / c-MET . After washing away unbound material, a biotinylated detection antibody is used to detect only phosphorylated receptor, utilizing a standard HRP format.
Product Specifications
Assay Format
96-well strip plate (sold separately)
Sample Volume Required
Cell lysates (100 µL)
Detection Method
Colorimetric ELISA - 450nm (TMB)
Conjugate
Biotin
Specificity
Label
HRP
Scientific Data Images for Human Phospho-HGFR/c-MET DuoSet IC ELISA
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).
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).
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.Kit Contents for Human Phospho-HGFR/c-MET DuoSet IC ELISA
- 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
Preparation and Storage
Shipping
The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.
Stability & Storage
Store the unopened product at 2 - 8 °C. Do not use past expiration date.
Background: HGFR/c-MET
HGFR (Hepatocyte Growth Factor Receptor), also known as Met, is a receptor tyrosine kinase that plays a central role in epithelial morphogenesis and cancer development. In the absence of ligand, HGFR forms noncovalent complexes with a variety of membrane proteins including CD44v6, CD151, EGFR, Fas, Integrin alpha 6 beta 4, Plexins B1, 2, 3, and MSPR/Ron. Ligation of one complex component triggers activation of the other, followed by cooperative signaling effects. Formation of some of these heteromeric complexes is a requirement for epithelial cell morphogenesis and tumor cell invasion. Paracrine induction of epithelial cell scattering and branching tubulogenesis results from the stimulation of HGFR on undifferentiated epithelium by HGF released from neighboring mesenchymal cells.
Long Name
Hepatocyte Growth Factor Receptor
Alternate Names
c-MET, cMET, HGF R, MET
Gene Symbol
MET
Additional HGFR/c-MET Products
Product Documents for Human Phospho-HGFR/c-MET DuoSet IC ELISA
Product Specific Notices for Human Phospho-HGFR/c-MET DuoSet IC ELISA
For research use only
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