Human Phospho-M-CSF R DuoSet IC ELISA
R&D Systems, part of Bio-Techne | Catalog # DYC3268-2
Key Product Details
Assay Type
Solid Phase Sandwich ELISA
Sample Type
Cell Lysates
Reactivity
Human
Human Phospho-M-CSF R DuoSet IC ELISA Features
- 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
Product Summary for Human Phospho-M-CSF R DuoSet IC ELISA
This DuoSet IC ELISA contains the basic components required for the development of sandwich ELISAs to measure tyrosine-phosphorylated human M-CSF R / CD115 in cell lysates. An immobilized capture antibody specific for M-CSF R / CD115 binds both phosphorylated and unphosphorylated M-CSF R / CD115 . After washing away unbound material, a biotinylated detection antibody is used to detect only phosphorylated receptor, utilizing a standard HRP format.
Product Specifications
Assay Format
96-well strip plate (sold separately)
Sample Volume Required
Cell lysates (100 µL)
Detection Method
Colorimetric ELISA - 450nm (TMB)
Conjugate
Biotin
Specificity
Label
HRP
Scientific Data Images for 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 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
betaTHP-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.Kit Contents for Human Phospho-M-CSF R DuoSet IC ELISA
- 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
Lysis Buffer*
IC Diluent*
Blocking Buffer*
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
Preparation and Storage
Shipping
The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.
Stability & Storage
Store the unopened product at 2 - 8 °C. Do not use past expiration date.
Background: M-CSF R/CD115
Macrophage Colony-Stimulating Factor Receptor (M-CSF R), also known as Colony Stimulating Factor 1 Receptor (CSF1 R) and CD115, is a cell-surface protein that serves as a receptor for the cytokines M-CSF and IL-34. It is a member of the type III subfamily of receptor tyrosine kinases. Human M-CSF R/CD115 is 972 amino acids (aa) in length with a predicted molecular weight of approximately 108 kDa, and shares 60% and 64% aa sequence identity with the mouse and rat orthologs, respectively. It is a type I membrane protein with a 19 aa signal peptide, a 493 aa extracellular domain (ECD) containing the ligand-binding domain and five immunoglobulin-like domains, a 25 aa transmembrane region, and a 435 aa cytoplasmic domain (ICD). M-CSF R/CD115 undergoes proteolytic processing at at least two different sites. TACE/ADAM17 cleaves M-CSF R/CD115 in its ECD, resulting in the shedding of the functional ligand-binding ECD. M-CSF R/CD115 can also be cleaved in its transmembrane region. This processing releases the ICD into the cytosol, where it moves into the nucleus and regulates gene transcription.
Ligand binding to M-CSF R/CD115 induces receptor homodimerization and transphosphorylation. The phosphorylated intracellular domain then binds to a multitude of proteins that influence cell proliferation, survival, and differentiation, and cytoskeletal reorganization. M-CSF R/CD115 is expressed primarily on cells of the monocyte/macrophage lineage, including microglia, dendritic cells, stem cells, and cells in the developing placenta. It is required for macrophage survival and regulates lineage decisions and maturation of these cells. Overactivation of M-CSF R/CD115 has been shown to lead to a malignant phenotype in different cell types. Additionally, it has been suggested that activated M-CSF R/CD115 is a predictor of poor outcome in several cancers including breast and ovarian. M-CSF R/CD115 expression has also been shown to be increased in microglia in brains afflicted with Alzheimer's disease and following injury.
Long Name
Macrophage Colony Stimulating Factor Receptor
Alternate Names
c-fms, CD115, CSF1R, M-CSFR, MCSFR
Gene Symbol
CSF1R
Additional M-CSF R/CD115 Products
Product Documents for Human Phospho-M-CSF R DuoSet IC ELISA
Product Specific Notices for Human Phospho-M-CSF R DuoSet IC ELISA
For research use only
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