Human Phospho-M-CSF R DuoSet IC ELISA
Human Phospho-M-CSF R DuoSet IC ELISA Summary
* Provided that the recommended microplates, buffers, diluents, substrates and solutions are used, and the assay is run as summarized in the Assay Procedure provided.
- Optimized capture and detection antibody pairings and recommended concentrations save lengthy development time
- Development protocols are provided to guide further assay optimization
- Assay can be customized to your specific needs
- Available in 2, 5, and 15-(96-well) plate pack sizes
- Economical alternative to Western blot
- Capture Antibody
- Conjugated Detection Antibody
- Calibrated Immunoassay Standard or Control
Other Reagents Required
PBS: (Catalog # DY006), or 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2O4, pH 7.2 - 7.4, 0.2 µm filtered
Wash Buffer: (Catalog # WA126), or equivalent
Substrate Solution: 1:1 mixture of Color Reagent A (H2O2) and Color Reagent B (Tetramethylbenzidine) (Catalog # DY999)
Stop Solution: 2 N H2SO4 (Catalog # DY994)
Microplates: R&D Systems (Catalog # DY990), or equivalent
Plate Sealers: ELISA Plate Sealers (Catalog # DY992), or equivalent
*For the Lysis Buffer, IC Diluent, and Blocking BUffer recommended for a specific DuoSet ELISA Development Kit, please see the product
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 WesternGloTM 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 ReceptorDuoSet 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.
Preparation and Storage
Background: M-CSF R/CD115
M-CSF R/CD115, the product of the c-fms proto-oncogene, is a member of the type III subfamily of receptor tyrosine kinases that also includes c-kit, the receptor for SCF, and the alpha and beta receptors for PDGF. M-CSF R/CD115 is expressed primarily on cells of the monocyte/macrophage lineage and in various tissues of the developing placenta.
Citation for Human Phospho-M-CSF R DuoSet IC ELISA
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
1 Citation: Showing 1 - 1
Multidimensional profiling of CSF1R screening hits and inhibitors: assessing cellular activity, target residence time, and selectivity in a higher throughput way.
Authors: Uitdehaag J, Sunnen C, van Doornmalen A, de Rouw N, Oubrie A, Azevedo R, Ziebell M, Nickbarg E, Karstens W, Ruygrok S
J Biomol Screen, 2011-08-26;16(9):1007-17.
Sample Types: Cell Lysates
Which phosphorylated sites are recognized by this assay?
This assay utilizes an anti-phosphorylated tyrosine monoclonal detection antibody, and it recognizes all phosphorylated tyrosine residues.
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