Figure 1. Treatment of H526 cells with SCF stimulates phosphorylation of c-Kit at Tyr719, detected by PathScan® Phospho-c-Kit (Tyr719) Sandwich ELISA Kit #7298, but does not affect the level of total c-Kit protein detected by PathScan® Total c-Kit Sandwich ELISA Kit #7197. The absorbance readings at 450 nm are shown in the top figure, while the corresponding Western blot using Phospho-c-Kit (Tyr719) Antibody #3391 (right panel) or c-Kit Antibody #3392 (left panel), is shown in the bottom figure.
Figure 2. The relationship between protein concentration of lysates from untreated and SCF-treated H526 lysates and the absorbance at 450 nm is shown. Cells (0.5x106 cells/ml) were serum starved overnight and then treated with Human Stem Cell Factor (hSCF) #8925 (100 ng/ml) for 5 min at 37°C, and then lysed.
|Product Includes||Volume||Solution Color|
|c-Kit Mouse mAb Coated Microwells||96 tests|
|Phospho-c-Kit (Tyr719) 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|
CST's PathScan® Phospho-c-Kit (Tyr719) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of c-Kit protein when phosphorylated at Tyr719. A c-Kit Mouse mAb has been coated onto the microwells. After incubation with cell lysates, both phospho- and nonphospho-c-Kit proteins are captured by the coated antibody. Following extensive washing, Phospho-c-Kit (Tyr719) Rabbit Antibody is added to detect the captured phospho-c-Kit protein. Anti-rabbit IgG HRP-linked Antibody #7074 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 c-Kit protein phosphorylated at Tyr719.
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
CST's PathScan® Phospho-c-Kit (Tyr719) Sandwich ELISA Kit #7298 detects endogenous levels of c-Kit protein when phosphorylated at Tyr719. A significant induction of phosphorylation of c-Kit at Tyr719 is detected in SCF-treated H526 cells using PathScan® Phospho-c-Kit (Tyr719) Sandwich ELISA Kit #7298 (Figure 1). The level of total c-Kit protein in either nonphospho or phospho lysate detected by PathScan® Total c-Kit Sandwich ELISA Kit #7197 or Western analysis remain unchanged (Figure 1). This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.
c-Kit is a member of the subfamily of receptor tyrosine kinases that includes PDGF, CSF-1, and FLT3/flk-2 receptors (1,2). It plays a critical role in activation and growth in a number of cell types including hematopoietic stem cells, mast cells, melanocytes, and germ cells (3). Upon binding with its stem cell factor (SCF) ligand, c-Kit undergoes dimerization/oligomerization and autophosphorylation. Activation of c-Kit results in the recruitment and tyrosine phosphorylation of downstream SH2-containing signaling components including PLCγ, the p85 subunit of PI3 kinase, SHP2, and CrkL (4). Molecular lesions that impair the kinase activity of c-Kit are associated with a variety of developmental disorders (5), and mutations that constitutively activate c-Kit can lead to pathogenesis of mastocytosis and gastrointestinal stromal tumors (6). Tyr719 is located in the kinase insert region of the catalytic domain. c-Kit phosphorylated at Tyr719 binds to the p85 subunit of PI3 kinase in vitro and in vivo (7).
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