Human Phospho-VEGFR2/KDR DuoSet IC ELISA

R&D Systems, part of Bio-Techne | Catalog # DYC1766-2

R&D Systems, part of Bio-Techne
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DYC1766-2
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DYC1766E
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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

View all VEGFR2/KDR/Flk-1 ELISA Kits »

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

Please see the product datasheet

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 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.

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

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

Alternate Names

CD309, Flk-1, Flk1, KDR, KRD1, Ly73, VEGF R2

Entrez Gene IDs

3791 (Human); 16542 (Mouse)

Gene Symbol

KDR

Additional VEGFR2/KDR/Flk-1 Products

Product Documents for Human Phospho-VEGFR2/KDR DuoSet IC ELISA

Product Datasheets

Certificate of Analysis

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Product Specific Notices for Human Phospho-VEGFR2/KDR DuoSet IC ELISA

For research use only

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