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 WesternGlo^TM 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.