Human Phospho-Tie-2 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.
- 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
- 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: 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
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Figure 1: The Human Phospho-Tie-2 DuoSet® IC ELISA is more sensitive than immunoprecipitation (IP)-Western Blot analysis. Human Tie-2 transfected BaF3 mouse pro-B cells (BaF3-hTie-2) were treated with 600 ng/mL Recombinant Human Angiopoietin-1 Protein (Catalog # 923-AN) for 7 minutes to induce tyrosine phosphorylation of Tie-2. Serial dilutions of lysates were analyzed by (A) IP-Western Blot and (B) this DuoSet® IC ELISA. IPs were done using an anti-Tie-2 monoclonal antibody and goat anti-mouse agarose. Immunoblots were incubated with a Biotinylated Anti-Phosphotyrosine Monoclonal Antibody (Catalog # BAM1676) to detect human phospho-Tie-2. Bands were visualized with Streptavidin-HRP (Catalog # DY998) followed by chemiluminescent detection. Human Phospho-Tie-2 can be detected in this DuoSet® IC ELISA by using approximately 10-20 times less lysate than is needed for a conventional IP-Western Blot.
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Figure 2: The Human Phospho-Tie-2 DuoSet® IC ELISA detects ligand-induced Tie-2 tyrosine phosphorylation. BaF3-hTie-2 cells were untreated or treated with 600 ng/mL recombinant human Ang-1 for seven minutes. ELISA and IP-Western Blot (inset) analyses were done using 50 μg and 200 μg of lysate, respectively. IP-Western Blots for phospho-Tie-2 (p-Tie-2) were done as described in Figure 1. Blots were stripped and total Tie-2 was detected using a Biotinylated Anti-Tie-2 Polyclonal Antibody (Catalog # BAF313).
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Figure 3: The specificity of the Human Phospho-Tie-2 DuoSet® IC ELISA is confirmed by receptor competition. BaF3-hTie-2 cells were treated with 600 ng/mL recombinant human (rh) Ang-1 for seven minutes. The indicated amounts of recombinant extracellular domains of rhTie-2/Fc Chimera (Catalog # 313-TI), rhTie-1/Fc Chimera (Catalog # 619-TI), rhVEGF R2/Fc Chimera (Catalog # 357-KD) or rhVEGF R3/Fc Chimera (Catalog # 349-F4) were added to 50 μg lysate and analyzed using this ELISA. Competition was observed only with recombinant human Tie-2.
Preparation and Storage
Tie-2, also known as Tek, is a transmembrane receptor tyrosine kinase (RTK) that functions as a receptor for Angiopoietin family proteins. It is expressed by embryonic and adult endothelial cells, hematopoietic stem cells, and a circulating population of proangiogenic monocytes. Tie-2 signaling is activated by Angiopoietins-1 and -4, while it can be either activated or inhibited by Angiopoietins-2 and -3. Tie-2 plays an important role in maintaining vascular integrity by mediating endothelial cell-smooth muscle cell communication and inhibiting endothelial cell apoptosis. It is also required for embryonic development of the endocardium. In addition, Tie-2 signaling mediates the quiescence of bone marrow stem cells in response to osteoblast-produced Angiopoietin-1. This quiescence is critical for maintaining an ongoing hematopoietic capability in the bone marrow.
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