Human Total FGFR4 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.
This DuoSet IC ELISA contains the basic components required for the development of sandwich ELISAs to measure total FGF R4 in cell lysates. An immobilized capture antibody specific for FGF R4 binds both phosphorylated and unphosphorylated FGF R4. After washing away unbound material, a biotinylated detection antibody is used to detect both phosphorylated and unphosphorylated protein, utilizing a standard Streptavidin-HRP format.
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- Optimized capture and detection antibody pairings with 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
- 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
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: From Costar EIA Plate (Costar Catalog # 2592) or 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
The Human Total FGF R4 DuoSet® IC ELISA is more sensitive than immunoprecipitation (IP)-Western blot analysis. Lysates prepared from MDA-MB-453 human breast cancer cells were serially diluted and analyzed by (A) IP-Western blot and (B) this DuoSet® IC ELISA. IPs were performed using an anti-FGF R4 monoclonal antibody and goat anti-mouse agarose. Immunoblots were incubated with a biotinylated anti-FGF R4 monoclonal antibody to detect total FGF R4. Bands were visualized by chemiluminescent detection. Human FGF R4 can be detected by this DuoSet IC ELISA by using approximately 2 to 4 times less lysate than is needed for a conventional IP-Western blot.
The Human Total FGF R4 DuoSet® IC ELISA measures the relative level of FGF R4. Lysates were prepared from MDA-MB-453 human breast cancer cells and HEK293 human embryonic kidney cells transfected with human FGF R4 (HEK293-hFGF R4). ELISA was performed using 100 μg of the MDA-MB-453 cell lysate and 5 μg of the HEK293-hFGF R4 cell lysate. IP-Western blot analyses were performed as described in Figure 1, using 400 μg of cell lysate.
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FGF activity is mediated by a family of type I transmembrane tyrosine kinases, which undergo dimerization and autophosphorylation after ligand binding. Five distinct genes encode closely related FGF receptors, FGF R1 through 5. FGF Rs contain three Ig-like domains and a stretch of acidic residues between the first and second Ig-like domains. FGF R1, 2, 3, and -4 have a cytoplasmic split tyrosine-kinase domain, but FGF R5 does not. Multiple forms of FGF R1, 2, and 3 are generated by alternative splicing
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