Product Pathways - PathScan ELISA
PathScan® Total c-Kit Sandwich ELISA Kit #7197
| Kit Includes | Volume | Solution Color |
|---|---|---|
| c-Kit Antibody coated microwells | 96 tests | |
| c-Kit Detection Ab | 11 milliliters | Green |
| Anti-mouse IgG HRP-linked Ab | 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 | 15 milliliters | Yellowish |
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® 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 #3392* 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 #3304* 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.
Specificity / Sensitivity
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.
Western Blotting
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).
Sandwich ELISA
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.
Sandwich ELISA
Figure 2: The relationship between protein concentration of lysates from untreated and SCF-treated H526 cells and kit assay optical density readings. After starvation, H526 cells were treated with SCF #9907 (40 ng/ml) for 5 min at 37?C, and then lysed.
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
Have you published research involving the use of our products? If so we'd love to hear about it. Please let us know!
