Human Phospho-MSPR/Ron DuoSet IC ELISA

The Human Phospho-MSP R/Ron DuoSet® IC ELISA is more sensitive than immunoprecipitation (IP)-Western Blot analysis. MDA-MB-453 human breast cancer cells were treated with 400 ng/mL recombinant human MSP (Catalog # 352-MS) for five minutes to induce tyrosine phosphorylation of MSP R. Serial dilutions of lysates were analyzed by (A) IP-Western Blot and (B) this DuoSet® IC ELISA. IPs were performed using an anti-MSP R monoclonal antibody and goat anti-mouse agarose. Immunoblots were incubated with a biotinylated anti-phosphotyrosine monoclonal antibody (Catalog # BAM1676) to detect phospho-MSP R. Bands were visualized with Streptavidin-HRP (Catalog # DY998) followed by chemiluminescent detection. Human phospho-MSP R can be detected in this DuoSet® IC ELISA by using approximately 6-12 times less lysate than is needed for a conventional IP-Western Blot.

The Human Phospho-MSP R/Ron DuoSet® IC ELISA detects ligand-induced MSP R tyrosine phosphorylation. MDA-MB-453 human breast cancer cells were untreated or treated with 400 ng/mL recombinant human MSP for five minutes. ELISA and IP-Western Blot (inset) analyses were performed using 100 μg and 400 μg of lysate, respectively. IP-Western Blots for phospho-MSP R (p-MSP R) were performed as described in Figure 1. Blots were stripped and total MSP R was detected using a Biotinylated Anti-MSP R Polyclonal Antibody (Catalog # BAF691).

The specificity of the Human Phospho-MSP R/Ron DuoSet® IC ELISA is confirmed by receptor competition. MDA-MB-453 human breast cancer cells were treated with 400 ng/mL recombinant human (rh) MSP for five minutes. The indicated amounts of recombinant extracellular domains of human MSP R, human HGF R/Fc Chimera (Catalog # 358-MT), human IGF-I sR (Catalog # 391-GR), or human EGF R (Catalog # 1095-ER) were added to 100 μg lysate and analyzed using this DuoSet® IC ELISA. Competition was observed only with recombinant human MSP R.