Product Pathways - PathScan ELISA
PathScan® Phospho-c-Kit (Tyr719) Sandwich ELISA Kit #7298
| Kit Includes | Volume | Solution Color |
|---|---|---|
| c-Kit Mouse mAb Coated Microwells | 96 tests | |
| Phospho-c-Kit (Tyr719) Rabbit Detection Ab | 11 milliliters | Green |
| Anti-rabbit IgG HRP-Linked Antibody | 11 milliliters | Red |
| TMB Substrate | 11 milliliters | Colorless |
| STOP Solution | 11 milliliters | Colorless |
| Sealing Tape | 2 sheets | |
| 20X Wash Buffer | 25 milliliters | Colorless |
| Sample Diluent | 25 milliliters | Blue |
| Cell Lysis Buffer (10X) # 9803 |
Note: 12 8-well modules –Each module is designed to break apart for 8 tests.
Note: Kit should be stored at 4°C with the exception of Cell Lysis Buffer (10X), which is stored at –20°C (packaged separately).
Species Cross-Reactivity
H
Reactivity Key: H=Human
Description
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.
Specificity / Sensitivity
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).
Sandwich ELISA
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 SCF #9907 (40 ng/ml) for 5 min at 37?C.
Sandwich ELISA
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.
Background
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 such as 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), while 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).
- Martin, F.H. et al. (1990) Cell 63, 203-211.
- Yarden, Y. et al. (1987) EMBO J. 6, 3341-3351.
- Gommerman, J.L. et al. (1997) J. Biol. Chem. 272, 30519-30525.
- Sattler, M. et al. (1997) J. Biol. Chem. 272, 10248-10253.
- Nocka, K. et al. (1990) EMBO J. 9, 1805-1813.
- Hirota, S. et al. (1998) Science 279, 577-580.
- Blume-Jensen, P. et al. (2000) Nat. Genet. 24, 157-162.
Application References
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Companion Products
- 3308 c-Kit (Ab81) Mouse mAb
- 3310 c-Kit (Ab81) Mouse mAb (Alexa Fluor® 488 Conjugate)
- 3391 Phospho-c-Kit (Tyr719) Antibody
- 3392 c-Kit Antibody
- 7074 Anti-rabbit IgG, HRP-linked Antibody
- 9803 Cell Lysis Buffer (10X)
- 7002 STOP Solution
- 7004 TMB Substrate
- 9808 Phosphate Buffered Saline (PBS-20X)
- 9809 Phosphate Buffered Saline with Tween 20 (PBST-20X)
- 9907 Human Stem Cell Factor (SCF)
