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.