Human Phospho-Tie-2 DuoSet IC ELISA
R&D Systems, part of Bio-Techne | Catalog # DYC2720-2
Key Product Details
Assay Type
Solid Phase Sandwich ELISA
Sample Type
Cell Lysates
Reactivity
Human
Human Phospho-Tie-2 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-Tie-2 DuoSet IC ELISA
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 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-Tie-2 DuoSet IC ELISA
Figure 1. The Human Phospho-Tie-2 DuoSet IC ELISA is more sensitive than immunoprecipitation (IP)-Western analysis
Human Tie-2 transfected Baf/3 cells (Baf/3-hTie-2) were treated with 600 ng/mL recombinant human Ang-1 for seven minutes to induce tyrosine phosphorylation of Tie-2. Serial dilutions of lysates were analyzed by (A) IP-Western blot and (B) this 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 phospho-Tie-2. Bands were visualized with Streptavidin-HRP (Cat # DY998) followed by chemiluminescent detection using WesternGloTM Chemiluminescent Detection Substrate (Catalog # AR004).
Figure 2. The Human Phospho-Tie-2 DuoSet IC ELISA detects ligand-induced Tie-2 tyrosine phosphorylation
Baf/3-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). Human Phospho-Tie-2 can be detected in this ELISA by using approximately 4 times less lysate than is needed for a conventional IP-Western blot.
Figure 3. The specificity of the Human Phospho-Tie-2 DuoSet IC ELISA is confirmed by receptor competition
Baf/3-hTie-2 cells were treated with 600 ng/mL recombinant human Ang-1 for seven minutes. The indicated amounts of recombinant extracellular domains of human Tie-2(Catalog #313-TI), human Tie-1 (Catalog #619-TI), human VEGF R2 (Catalog #357-KD) or human VEGF R3 (Catalog #321-FL) were added to 50 μg lysate and analyzed using this ELISA. Competition was observed only with recombinant Tie-2.Kit Contents for Human Phospho-Tie-2 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: Tie-2
Long Name
Tyrosine Kinase with Immunoglobulin and Epidermal Growth Factor Homology Domains 2
Alternate Names
CD202b, TEK, Tie2
Entrez Gene IDs
Gene Symbol
TEK
Additional Tie-2 Products
Product Documents for Human Phospho-Tie-2 DuoSet IC ELISA
Product Specific Notices for Human Phospho-Tie-2 DuoSet IC ELISA
For research use only
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