Human Phospho-VEGFR2/KDR DuoSet IC ELISA
R&D Systems, part of Bio-Techne | Catalog # DYC1766-2
Key Product Details
Assay Type
Solid Phase Sandwich ELISA
Sample Type
Cell Lysates
Reactivity
Human
Human Phospho-VEGFR2/KDR 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-VEGFR2/KDR DuoSet IC ELISA
This DuoSet IC ELISA contains the basic components required for the development of sandwich ELISAs to measure tyrosine-phosphorylated human VEGF R2 / KDR / Flk-1 in cell lysates. An immobilized capture antibody specific for VEGF R2 / KDR / Flk-1 binds both phosphorylated and unphosphorylated VEGF R2 / KDR / Flk-1 . 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-VEGFR2/KDR DuoSet IC ELISA
Figure 1. The Human Phospho-VEGF R2 DuoSet IC ELISA is more sensitive than immunoprecipiation (IP)-Western blot analysis
Human umbilical vein endothelial cells (HUVEC) were treated with 100 ng/mL recombinant human VEGF165 (Catalog # 293-VE) for five minutes to induce tyrosine phosphorylation of VEGF R2. Dilutions of HUVEC lysates were analyzed by this ELISA (Catalog # DYC1766) and IP-Western blot (inset). IPs were performed using an anti-VEGF R2 monoclonal antibody and anti-mouse IgG agarose. Immunoblots were incubated with a biotinylated anti-phospho-tyrosine monoclonal antibody (Catalog # BAM1676) to detect phospho-VEGF R2 (p-VEGF R2). Bands were visualized with Streptavidin-HRP (Catalog # DY998) followed by chemiluminescent detection. Blots were stripped and total VEGF R2 (VEGF R2) was detected using a biotinylated polyclonal anti-VEGF R2 antibody (Catalog # BAF357).
Figure 2. The Human Phospho-VEGF R2DuoSet IC ELISA detects ligand-induced VEGF R2 tyrosine phosphorylation
HUVECs were untreated or treated with 100 ng/mL recombinant human VEGF165 for five minutes. ELISA and IP-Western blot (inset) analyses were performed using 100 μg and 1000 μg of lysate, respectively. IP-Western blots were performed as described in Figure 1.
Figure 3. The specificity of the Human Phospho-VEGF R2DuoSet IC ELISA is confirmed by receptor competition
HUVECs were treated with 100 ng/mL of human recombinant VEGF165 for five minutes. The indicated amounts of extracellular domains of recombinant VEGF R2 (Catalog # 357-KD), VEGF R1 (Catalog # 321-FL) or VEGF R3 (Catalog # 349-F4) were added to 100 μg lysate and analyzed by this ELISA. Competition was observed only with recombinant VEGF R2.Kit Contents for Human Phospho-VEGFR2/KDR 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: VEGFR2/KDR/Flk-1
VEGFR2/KDR/Flk-1 (vascular endothelial growth factor receptor 2) is a transmembrane receptor tyrosine kinase that mediates the angiogenic effects of VEGF-A and VEGF-C. It is expressed primarily on vascular endothelial cells and endothelial cell progenitors. It is also expressed on endometrial epithelium, hematopoietic stem cells, liver sinusoidal endothelial cells, Sertoli cells and Leydig cells, platelets and megakaryocytes, sensory and autonomic neurons, Schwann cells, Muller glial cells, retinal progenitors, and osteoblasts. VEGFR2 can associate with VEGFR1, and a soluble form of VEGFR2 can circulate in the serum.
Long Name
Vascular Endothelial Growth Factor Receptor 2
Alternate Names
CD309, Flk-1, Flk1, KDR, KRD1, Ly73, VEGF R2
Gene Symbol
KDR
Additional VEGFR2/KDR/Flk-1 Products
Product Documents for Human Phospho-VEGFR2/KDR DuoSet IC ELISA
Product Specific Notices for Human Phospho-VEGFR2/KDR DuoSet IC ELISA
For research use only
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