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 (Tyr699) Sandwich ELISA Kit #12946, 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) and Phospho-M-CSF Receptor (Tyr699) (D10B11) Rabbit mAb #12251 (right) 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® Total M-CSF Receptor 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|
|M-CSF Receptor Rabbit Detection Antibody||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|
PathScan® Total M-CSF Receptor Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of M-CSF receptor protein. An M-CSF receptor mouse mAb has been coated onto the microwells. After incubation with cell lysates, both phospho- and nonphospho-M-CSF receptor proteins are captured by the coated antibody. Following extensive washing, a M-CSF receptor rabbit antibody is added to detect both the captured phospho- and nonphospho-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 optical density for this developed color is proportional to the quantity of M-CSF receptor protein.
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® Total M-CSF Receptor Sandwich ELISA Kit detects endogenous levels of 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.
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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