Figure 1. Treatment of H526 cells with SCF stimulates tyrosine phosphorylation of c-Kit, detected by PathScan® Phospho-c-Kit (panTyr) Sandwich ELISA Kit #7231, 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.
Figure 3. Kit specificity as demonstrated by Western analysis of the ELISA microwell captured protein. Lysates were prepared from untreated and SCF-treated H526 cells and incubated in microwells coated with the c-Kit capture antibody. Wells were washed and the captured protein was solubilized in SDS gel loading buffer. Western analysis of untreated and SCF-treated H526 cell starting lysates (lanes 1 & 2) and the captured protein (lanes 3 & 4) was carried out using Phospho-Tyrosine Mouse mAb (P-Tyr-100) (Biotinylated) #9417. The major band detected in the captured material corresponds to the phospho-c-Kit protein (lane 4).
The PathScan® Phospho-c-Kit (panTyr) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of c-Kit when tyrosine phosphorylated. A c-Kit Mouse mAb has been coated onto the microwells. After incubation with cell lysates, c-Kit (phospho and nonphospho) is captured by the coated antibody. Following extensive washing, a Biotinylated Phospho-Tyrosine Mouse Detection Antibody is added to detect tyrosine phosphorylation of the captured c-Kit protein. HRP-linked Strepavidin is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of the absorbance for this developed color is proportional to the quantity of c-Kit phosphorylated on tyrosine.
Antibodies in kit are custom formulations specific to kit.
CST's PathScan® Phospho-c-Kit (panTyr) Sandwich ELISA Kit #7231 detects c-Kit when tyrosine phosphorylated. As shown in Figure 1, a significant induction of c-Kit tyrosine phosphorylation can be detected in H526 cells following treatment with SCF using the c-Kit (panTyr) Sandwich ELISA Kit #7231. The level of total c-Kit (phospho and nonphospho) remains unchanged as shown by Western analysis and by PathScan® Total c-Kit Sandwich ELISA Kit #7197. In Figure 3, Western blot analysis of protein captured in the c-Kit antibody coated microwell shows major band corresponding to the phospho-c-Kit protein. 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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PathScan is a trademark of Cell Signaling Technology, Inc.