Human Phospho-Axl DuoSet IC ELISA

Figure 1. The Human Phospho-Axl DuoSet IC ELISA is more sensitive than immunoprecipitation (IP)-Western analysis Human glioblastoma cell line, A172, was treated with conditioned media from the human lung cell line, WI38, for fifteen minutes to induce tyrosine phosphorylation of Axl. Serial dilutions of lysates were analyzed by (A) IP-Western blot and (B) this ELISA. IPs were done using an anti-Axl monoclonal antibody and anti-mouse IgG agarose. Immunoblots were incubated with a biotinylated anti-phosphotyrosine monoclonal antibody (Catalog # BAM1676) to detect phospho-Axl (p-Axl). Bands were visualized with Streptavidin-HRP (Cat # DY998) followed by chemiluminescent detection.

Figure 2. The Human Phospho-AxlDuoSet IC ELISA detects ligand-induced Axl tyrosine phosphorylation A172 cells were untreated or treated with conditioned media from the human lung cell line, WI38, for fifteen minutes. ELISA and IP-Western blot (inset) analyses were done using 100 μg and 400 μg of lysate, respectively. IP-Western blots for phospho-Axl (p-Axl) were done as described in Figure 1. Blots were stripped and total Axl was detected using a biotinylated anti-Axl polyclonal antibody (Catalog # BAF154).

Figure 3. The specificity of the Human Phospho-AxlDuoSet IC ELISA is confirmed by receptor competition A172 cells were treated with conditioned media from the human lung cell line WI38 for fifteen minutes. The indicated amounts of recombinant extracellular domains of human Axl (Catalog # 154-AL), human Dtk (Catalog # 859-DK) or human Trk A (Catalog # 175-TK) were added to 125 μg lysate and analyzed using this ELISA. Competition was observed only with recombinant Axl.