Human Phospho-Tie-2 DuoSet IC ELISA

Catalog # Availability Size / Price Qty
DYC2720E
DYC2720-5
DYC2720-2
Product Details
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Human Phospho-Tie-2 DuoSet IC ELISA Summary

Assay Type
Solid Phase Sandwich ELISA
Format
96-well strip plate
Sample Volume Required
Cell lysates (100 µL)
Sufficient Materials
Kits available for two, five, or fifteen 96-well plates*
Specificity
Please see the product datasheet

* 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 tyrosine-phosphorylated human Tie-2 in cell lysates. An immobilized capture antibody specific for Tie-2 binds both phosphorylated and unphosphorylated Tie-2. After washing away unbound material, a biotinylated detection antibody is used to detect only phosphorylated receptor, utilizing a standard HRP format.
 

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

Data
DYC2720 DuoSet IC ELISA Figure 1
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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.

DYC2720 DuoSet IC ELISA Figure 2
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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).

DYC2720 DuoSet IC ELISA Figure 3
View Larger Image

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.

Product Datasheets

Preparation and Storage

Stability & Storage
Store the unopened product at 2 - 8 °C. Do not use past expiration date.

Background: Tie-2

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.

Long Name:
Tyrosine Kinase with Immunoglobulin and Epidermal Growth Factor Homology Domains 2
Entrez Gene IDs:
7010 (Human); 21687 (Mouse); 30747 (Zebrafish)
Alternate Names:
angiopoietin-1 receptor; CD202b antigen; CD202b; EC 2.7.10; EC 2.7.10.1; hTIE2; p140 TEK; soluble TIE2 variant 1; soluble TIE2 variant 2; TEK tyrosine kinase, endothelial; TEK; Tie2; Tie-2; TIE2CD202b; Tunica interna endothelial cell kinase; Tyrosine-protein kinase receptor TEK; Tyrosine-protein kinase receptor TIE-2; venous malformations, multiple cutaneous and mucosal; VMCM; VMCM1

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