Figure 1: Treatment of NKM-1 cells with Human Macrophage Colony Stimulating Factor (hM-CSF) #8929 stimulates tyrosine phosphorylation of M-CSF receptor protein, as detected by PathScan® Phospho-M-CSF Receptor (panTyr) Sandwich ELISA Kit, but does not affect the level of total M-CSF receptor detected by PathScan® Total M-CSF Receptor Sandwich ELISA Kit #13032. The absorbance readings at 450 nm are shown in the top figure while corresponding western blots using M-CSF Receptor Antibody #3152 (left panel) and Phospho-M-CSF Receptor (Tyr699) (D10B11) Rabbit mAb #12251 (right panel) are shown in the bottom figure.
Figure 2: The relationship between protein concentration of lysates from untreated and hM-CSF-treated NKM-1 cells and the absorbance at 450 nm as detected by the PathScan® Phospho-M-CSF Receptor (panTyr) Sandwich ELISA Kit is shown. Unstarved NKM-1 cells (0.5-1.0 x 106) were treated with Human Macrophage Colony Stimulating Factor (hM-CSF) #8929 (50 ng/ml) for 2-5 min at 37°C and then lysed.
|Product Includes||Volume||Solution Color|
|M-CSFR Mouse mAb Coated Microwells||96 tests|
|Phospho Tyrosine Rabbit Detection mAb (7G11)||1 ea||Green (Lyophilized)|
|Anti-rabbit IgG, HRP-linked Antibody (ELISA Formulated)||1 ea||Red (Lyophilized)|
|Detection Antibody Diluent||11 ml||Green|
|HRP Diluent||11 ml||Red|
|TMB Substrate 7004||11 ml|
|STOP Solution 7002||11 ml|
|Sealing Tape||2 ea|
|ELISA Wash Buffer (20X) 9801||25 ml|
|ELISA Sample Diluent||25 ml||Blue|
|Cell Lysis Buffer (10X) 9803||15 ml|
The PathScan® Phospho-M-CSF Receptor (panTyr) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of tyrosine-phosphorylated M-CSF receptor protein. A M-CSF Receptor Mouse mAb has been coated onto the microwells. After incubation with cell lysates, M-CSF receptor protein (phospho and nonphospho) is captured by the coated antibody. Following extensive washing, a phospho-tyrosine rabbit detection mAb is added to detect the captured phospho-M-CSF receptor proteins. Anti-rabbit IgG, HRP-linked antibody is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of absorbance for this developed color is proportional to the quantity of M-CSF receptor protein phosphorylated on tyrosine residues.
Antibodies in kit are custom formulations specific to kit.
NOTE: Prepare solutions with purified water.
*NOTE: Some PathScan® ELISA Kits may include HRP-Linked Streptavidin in place of HRP-Linked Antibody.
NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP Solution.
posted November 2013
Protocol Id: 204
PathScan® Phospho-M-CSF Receptor (panTyr) Sandwich ELISA Kit recognizes endogenous levels of tyrosine-phosphorylated M-CSF receptor protein in human cells, as shown in Figure 1. The kit sensitivity is shown in Figure 2. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.Species Reactivity:
Macrophage-colony stimulating factor (M-CSF, CSF-1) receptor is an integral membrane tyrosine kinase encoded by the c-fms proto-oncogene. M-CSF receptor is expressed in monocytes (macrophages and their progenitors) and drives growth and development of this blood cell lineage. (1-3). Binding of M-CSF to its receptor induces receptor dimerization, activation, and autophosphorylation of cytoplasmic tyrosine residues used as docking sites for SH2-containing signaling proteins (4). There are at least five major tyrosine autophosphorylation sites. Tyr723 (Tyr721 in mouse) is located in the kinase insert (KI) region. Phosphorylated Tyr723 binds the p85 subunit of PI3 kinase as well as PLCγ2 (5). Phosphorylation of Tyr809 provides a docking site for Shc (5). Overactivation of this receptor can lead to a malignant phenotype in various cell systems (6). The activated M-CSF receptor has been shown to be a predictor of poor outcome in advanced epithelial ovarian carcinoma (7) and breast cancer (8).
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Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
PathScan is a trademark of Cell Signaling Technology, Inc.