Human Phospho-Mer DuoSet IC ELISA

The Human Phospho-Mer DuoSet IC ELISA is more sensitive than immunoprecipitation (IP)-Western analysis NS0 cells transfected with human Mer (NS0-hMer) were treated with conditioned media from the human lung cell line, WI-38, for 15 minutes to induce tyrosine phosphorylation of Mer. Lysates were serially diluted and analyzed by (A) IP-Western blot and (B) this DuoSet IC ELISA. IPs were done using an anti-Mer monoclonal antibody and goat anti-mouse agarose. Immunoblots were incubated with an HRP-conjugated anti-phosphotyrosine monoclonal antibody (R&D Systems, Catalog # HAM1676) to detect phospho-Mer. Bands were visualized by chemiluminescent detection using WesternGlo^TM Chemiluminescent Detection Substrate (R&D Systems, Catalog # AR004). Human Phospho-Mer can be detected in this DuoSet IC ELISA by using approximately 5 to 10 times less lysate than is needed for a conventional IP-Western blot.

The Human Phospho-MerDuoSet IC ELISA detects ligand-induced Mer tyrosine phosphorylation NS0-hMer cells were untreated or treated with conditioned media from the human lung cancer cell line, WI-38, 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-Mer (p-Mer) were done as described in Figure 1. Blots were stripped and total Mer was detected using a biotinylated anti-Mer polyclonal antibody (R&D Systems, Catalog #BAF891).

The specificity of the Human Phospho-MerDuoSet IC ELISA is confirmed by receptor competition NS0-hMer cells were treated with conditioned media from the human lung cell line, WI-38, for 15 minutes minutes. The indicated amounts of recombinant extracellular domains of human Mer/Fc (R&D Systems, Catalog #891-MR), human Axl/Fc (R&D Systems, Catalog #154-AL), human Dtk/Fc (R&D Systems, Catalog #859-DK) or human TrkA/Fc (R&D Systems, Catalog #175-TK) were added to 100 μg lysate and analyzed using this DuoSet IC ELISA. Competition was observed only with recombinant human Mer.