Human Phospho-Histone H2AX (S139) DuoSet IC ELISA

Standard Curve

The Human Phospho-Axl DuoSet IC ELISA is more sensitive than immunoprecipitation (IP)-Western blot analysis. A172 human glioblastoma cells were treated with Gas-6-containing conditioned media1 from the human lung cell line, WI38, for fifteen minutes to induce tyrosine phosphorylation of Axl. Lysates were serially diluted and analyzed by (A) IP-Western blot and (B) this DuoSet IC ELISA. IPs were done using an Anti-Human Axl Monoclonal Antibody and Anti-Mouse IgG Agarose. Immunoblots were incubated with a Biotinylated Anti-Human Phospho-tyrosine Monoclonal Antibody (Catalog # BAM1676) to detect phospho-Axl. Bands were visualized with Streptavidin-HRP (Catalog # DY998) followed by chemiluminescent detection.

The Human Phospho-Axl DuoSet IC ELISA detects ligand-induced Axl tyrosine phosphorylation. A172 cells were untreated or treated with Gas-6-containing conditioned media1 from the human lung cell line, WI38, for 15 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 (Axl) was detected using a Biotinylated Anti-Human Axl Polyclonal Antibody (Catalog # BAF154).

The specificity of the Human Phospho-Axl DuoSet IC ELISA is confirmed by receptor competition. A172 cells were treated with Gas-6-containing conditioned media1 from the human lung cell line, WI38, for 15 minutes. The indicated amounts of recombinant extracellular domains of human Axl/Fc Chimera (Catalog # 154-AL), human Dtk/Fc Chimera (Catalog # 859-DK) or human TrkA/Fc Chimera (Catalog # 175-TK) were added to 125 µg of lysate and analyzed using this DuoSet IC ELISA. Competition was observed only with recombinant human Axl.