Human Phospho-M-CSF R DuoSet IC ELISA

Figure 1. The Human Phospho-M-CSF Receptor DuoSet IC ELISA is more sensitive than immunoprecipitation (IP)-Western analysis The human acute monocytic leukemia cell line THP-1 was treated with 600 ng/mL recombinant human M-CSF (R&D Systems, Catalog #216-MC) for five minutes to induce tyrosine phosphorylation of M-CSF Receptor. Lysates were serially diluted and analyzed by (A) IP-Western blot and (B) this DuoSet IC ELISA. IPs were done using an anti-M-CSF Receptor polyclonal antibody and protein G agarose. Immunoblots were incubated with a biotinylated anti-phosphotyrosine monoclonal antibody (R&D Systems, Catalog # BAM1676) to detect phospho-M-CSF Receptor. Bands were visualized with Streptavidin-HRP (R&D Systems, Cat # DY998) followed by chemiluminescent detection using WesternGlo^TM Chemiluminescent Detection Substrate (R&D Systems, Catalog # AR004). Human Phospho-M-CSF Receptor can be detected in this DuoSet IC ELISA by using approximately 4 to 8 times less lysate than is needed for a conventional IP-Western blot.

Figure 2. The Human Phospho-M-CSF Receptor DuoSet IC ELISA detects ligand-induced Flt-3 tyrosine phosphorylation THP-1 cells were untreated or treated with 600 ng/mL recombinant human M-CSF for five minutes. ELISA and IP-Western blot (inset) analyses were done using 100 μg and 400 μg of lysate, respectively. IP-Western blots for phospho-M-CSF Receptor (p-M-CSF R) were done as described in Figure 1. Blots were stripped and total M-CSF Receptor was detected using a biotinylated anti-M-CSF Receptor polyclonal antibody (R&D Systems, Catalog #BAF329).

Figure 3. The specificity of the Human Phospho-M-CSF ReceptorDuoSet IC ELISA is confirmed by receptor competition beta THP-1 cells were treated with 600 ng/mL recombinant human M-CSF for five minutes. The indicated amounts of recombinant extracellular domains of human M-CSF Receptor (R&D Systems, Catalog #329-MR), human SCF sR/c-kit (R&D Systems, Catalog #332-SR), human Flt-3 (R&D Systems, Catalog# 368-ST), human PDGF sR alpha (R&D Systems, Catalog #322-PR), or human PDGF R beta (R&D Systems, Catalog #385-PR) were added to 100 μg lysate and analyzed using this DuoSet IC ELISA. Competition was observed only with recombinant Human M-CSF Receptor.