Figure 1: Non-phospho and phospho c-Kit protein from untreated and SCF-treated H526 cells detected by PathScan® Total c-Kit Sandwich ELISA kit #7197 with similar optical density readings. OD 450 readings 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 demonstrated by Western blot analysis of the ELISA-well captured protein. Lysates prepared from human H526 cells were incubated in wells coated with capture antibody #3392. Wells were washed and captured protein was solubilized in SDS gel loading buffer. H526 starting lysate (lane 1) and captured protein (lane 2) were analyzed by Western blot using c-Kit antibody #3304. A single band corresponding to the c-Kit protein is detected in the captured material (lane 2).
CST's PathScan® Total c-Kit Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of total c-Kit protein. A c-Kit Antibody 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, c-Kit Mouse mAb is added to detect both the captured both phospho- and nonphospho-c-Kit proteins. Anti-mouse IgG, HRP-linked Antibody #7076* 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 total c-Kit protein.
* Antibodies in kit are custom formulations specific to kit.
CST's PathScan® Total c-Kit Sandwich ELISA Kit #7197 detects endogenous levels of total c-Kit protein. As shown in Figure 1, PathScan® Total c-Kit Sandwich ELISA Kit #7197 detects both phospho and non-phospho c-Kit protein from untreated and SCF-treated H526 cell lysates. In Figure 3, Western blot analysis of protein captured in the c-Kit Antibody #3392 coated microwell shows a single band corresponding to the 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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Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
PathScan is a trademark of Cell Signaling Technology, Inc.