Human Phospho-VEGFR2/KDR DuoSet IC ELISA

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Ancillary Products Available
Figure 1. The Human Phospho-VEGF R2 DuoSet IC ELISA is more sensitive than immunoprecipiation (IP)-Western blot analysis
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Product Details
Citations (11)
Supplemental Products

Human Phospho-VEGFR2/KDR DuoSet IC ELISA Summary

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

Scientific Data

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 R2DuoSet 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 R2DuoSet 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.

Product Datasheets

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Preparation and Storage

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

VEGF R1 (Flt-1), VEGF R2 (KDR/Flk-1), and VEGF R3 (Flt-4) belong to the class III subfamily of receptor tyrosine kinases (RTKs). All three receptors contain seven immunoglobulin-like repeats in their extracellular domain and kinase insert domains in their intracellular region. They are best known for regulating VEGF family-mediated vasculogenesis, angiogenesis, and lymphangiogenesis. They are also mediators of neurotrophic activity and regulators of hematopoietic development. VEGF R2 is thought to be the primary inducer of VEGF-mediated blood vessel growth, while VEGF R3 plays a significant role in VEGF-C and VEGF-D-mediated lymphangiogenesis.

Long Name:
Vascular Endothelial Growth Factor Receptor 2
Entrez Gene IDs:
3791 (Human); 16542 (Mouse)
Alternate Names:
CD309 antigen; CD309; EC 2.7.10; EC; Fetal liver kinase 1; fetal liver kinase-1; Flk1; Flk-1; FLK1tyrosine kinase growth factor receptor; KDR; kinase insert domain receptor (a type III receptor tyrosine kinase); Kinase insert domain receptor; KRD1; Ly73; Protein-tyrosine kinase receptor flk-1; soluble VEGFR2; vascular endothelial growth factor receptor 2; VEGF R2; VEGFR; VEGFR2; VEGFR-2

Citations for Human Phospho-VEGFR2/KDR DuoSet IC ELISA

R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.

11 Citations: Showing 1 - 10
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  1. Dual Action of Sulfated Hyaluronan on Angiogenic Processes in Relation to Vascular Endothelial Growth Factor-A
    Authors: L Koehler, G Ruiz-Gómez, K Balamuruga, S Rother, J Freyse, S Möller, M Schnabelra, S Köhling, S Djordjevic, D Scharnwebe, J Rademann, MT Pisabarro, V Hintze
    Sci Rep, 2019;9(1):18143.
    Species: Human
    Sample Types: Cell Lysates
  2. Combinations of Bevacizumab and Erlotinib show activity in colorectal cancer independent of RAS status
    Authors: P Mésange, A Bouygues, N Ferrand, M Sabbah, AE Escargueil, A Savina, B Chibaudel, C Tournigand, T Andre, A de Gramont, AK Larsen
    Clin. Cancer Res., 2018;0(0):.
    Species: Human
    Sample Types: Whole Cells
  3. Andrographolide enhanced 5-fluorouracil-induced antitumor effect in colorectal cancer via inhibition of c-MET pathway
    Authors: M Su, B Qin, F Liu, Y Chen, R Zhang
    Drug Des Devel Ther, 2017;11(0):3333-3341.
    Species: Human
    Sample Types: Cell Culture Supernates
  4. Modulation of VEGF signaling in a mouse model of diabetes by xanthohumol and 8-prenylnaringenin: Unveiling the angiogenic paradox and metabolism interplay
    Authors: Raquel Costa
    Mol Nutr Food Res, 2016;0(0):.
    Species: Human
    Sample Types: Cell Lysates
  5. The propeptides of VEGF-D determine heparin binding, receptor heterodimerization, and effects on tumor biology.
    Authors: Harris, Nicole C, Davydova, Natalia, Roufail, Sally, Paquet-Fifield, Sophie, Paavonen, Karri, Karnezis, Tara, Zhang, You-Fang, Sato, Teruhiko, Rothacker, Julie, Nice, Edouard, Stacker, Steven A, Achen, Marc G
    J Biol Chem, 2013;288(12):8176-86.
    Species: Human
    Sample Types: Cell Lysates
  6. AZD4547: an orally bioavailable, potent, and selective inhibitor of the fibroblast growth factor receptor tyrosine kinase family.
    Authors: Gavine PR, Mooney L, Kilgour E
    Cancer Res., 2012;72(8):2045-56.
    Species: Human
    Sample Types: Cell Lysates
  7. Biomarkers for antitumor activity of bevacizumab in gastric cancer models.
    Authors: Yamashita-Kashima Y, Fujimoto-Ouchi K, Yorozu K, Kurasawa M, Yanagisawa M, Yasuno H, Mori K
    BMC Cancer, 2012;12(0):37.
    Species: Human
    Sample Types: Cell Lysates
  8. The 12th-14th type III repeats of fibronectin function as a highly promiscuous growth factor-binding domain.
    Authors: Martino MM, Hubbell JA
    FASEB J., 2010;24(12):4711-21.
    Species: Human
    Sample Types: Cell Lysates
  9. Neuropilin-2 mediates VEGF-C-induced lymphatic sprouting together with VEGFR3.
    Authors: Xu Y, Yuan L, Mak J, Pardanaud L, Caunt M, Kasman I, Larrivee B, Del Toro R, Suchting S, Medvinsky A, Silva J, Yang J, Thomas JL, Koch AW, Alitalo K, Eichmann A, Bagri A
    J. Cell Biol., 2010;188(1):115-30.
    Species: Human
    Sample Types: Cell Lysates
  10. Neuropilin-1 binds to VEGF121 and regulates endothelial cell migration and sprouting.
    Authors: Pan Q, Chathery Y, Wu Y, Rathore N, Tong RK, Peale F, Bagri A, Tessier-Lavigne M, Koch AW, Watts RJ
    J. Biol. Chem., 2007;282(33):24049-56.
    Species: Human
    Sample Types: Cell Lysates
  11. Neuropilin-2 interacts with VEGFR-2 and VEGFR-3 and promotes human endothelial cell survival and migration.
    Authors: Favier B, Alam A, Barron P, Bonnin J, Laboudie P, Fons P, Mandron M, Herault JP, Neufeld G, Savi P, Herbert JM, Bono F
    Blood, 2006;108(4):1243-50.
    Species: Human
    Sample Types: Cell Lysates


  1. Which phosphorylated sites are recognized by this assay?

    • This assay utilizes an anti-phosphorylated tyrosine monoclonal detection antibody, and it recognizes all phosphorylated tyrosine residues.

  2. Is it possible to obtain quantitative data from Human Phospho-VEGFR2/KDR DuoSet IC ELISA?

    • Human Phospho-VEGFR2/KDR DuoSet IC ELISA is a developmental ELISA designed to be semi-quantitative for detecting relative changes in sample phosphorylation. End users can optimize this kit to be quantitative. It is possible to construct a standard curve from the control provided in the kit. The linear bounds would need to be empirically verified by the end user as R&D Systems has not tested this optimization in-house.

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