Product Pathways - PathScan ELISA
PathScan® Phospho-c-Kit (panTyr) Sandwich ELISA Kit #7231
|7231C||1 Kit (96 assays)||---||In Stock||---|
|7231||carrier free and custom formulation / quantity||email request|
When ordering five or more kits, please contact us for processing time and pricing at firstname.lastname@example.org.
|Kit Includes||Volume||Solution Color|
|c-Kit mAb coated microwells||1 Ea|
|Phospho Tyrosine Mouse Detection mAb (Biotinylated)||1 Ea||Green (Lyophilized)|
|HRP-Linked Streptavidin (ELISA Formulated)||1 Ea||Red (Lyophilized)|
|Detection Antibody Diluent||11 ml||Green|
|HRP Diluent||11 ml||Red|
|TMB Substrate #7004||11 ml||Colorless|
|STOP Solution #7002||11 ml||Colorless|
|ELISA Wash Buffer (20X)||25 ml||Colorless|
|ELISA Sample Diluent||25 ml||Blue|
|Cell Lysis Buffer (10X) #9803||15 ml|
|Sealing Tape||2 sheets|
Note: 12 8-well modules – Each module is designed to break apart for 8 tests.
Storage: Kit should be stored at 4°C with the exception of Lysis Buffer, which is stored at –20°C (packaged separately).
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.
Specificity / Sensitivity
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.
ELISA - Western correlation
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).
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).
- Martin, F.H. et al. (1990) Cell 63, 203-11.
- Yarden, Y. et al. (1987) EMBO J 6, 3341-51.
- Gommerman, J.L. et al. (1997) J Biol Chem 272, 30519-25.
- Sattler, M. et al. (1997) J Biol Chem 272, 10248-53.
- Nocka, K. et al. (1990) EMBO J 9, 1805-13.
- Hirota, S. et al. (1998) Science 279, 577-80.
- Blume-Jensen, P. et al. (2000) Nat Genet 24, 157-62.
Have you published research involving the use of our products? If so we'd love to hear about it. Please let us know!
- 9803 Cell Lysis Buffer (10X)
- 8925 Human Stem Cell Factor (hSCF)
- 7298 PathScan® Phospho-c-Kit (Tyr719) Sandwich ELISA Kit
- 7197 PathScan® Total c-Kit Sandwich ELISA Kit
- 9808 Phosphate Buffered Saline (PBS-20X)
- 9809 Phosphate Buffered Saline with Tween® 20 (PBST-20X)
- 9411 Phospho-Tyrosine Mouse mAb (P-Tyr-100)
- 9417 Phospho-Tyrosine Mouse mAb (P-Tyr-100) (Biotinylated)
- 9416 Phospho-Tyrosine Mouse mAb (P-Tyr-102)
- 3391 Phospho-c-Kit (Tyr719) Antibody
- 7002 STOP Solution
- 7004 TMB Substrate
- 3308 c-Kit (Ab81) Mouse mAb
- 3074 c-Kit (D13A2) XP® Rabbit mAb
This product is intended for research purposes only. The product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.
PathScan is a trademark of Cell Signaling Technology, Inc.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.