Cell Signaling Technology

Product Pathways - PathScan ELISA

PathScan® Phospho-c-Kit (Tyr719) Sandwich ELISA Kit #7298

When ordering five or more kits, please contact us for processing time and pricing at sales@cellsignal.com.

Important Ordering Details: Product is assembled upon order to ensure maximum activity. Domestic: Please allow up to two weeks for your order to be processed and shipped. International: Please allow up to three weeks, depending on the country, for your order to be processed and shipped.

Kit Includes Volume Solution Color
c-Kit mAb Coated Microwells 96 tests
P-c-Kit (Y719) Detection Ab 11 ml Green
Anti-rabbit IgG, HRP-linked Antibody 11 ml Red
TMB Substrate #7004 11 ml Colorless
STOP Solution #7002 11 ml Colorless
Sealing Tape 2 sheets
ELISA Wash Buffer (20X) 25 ml Colorless
ELISA Sample Diluent 25 ml Blue
Cell Lysis Buffer (10X) #9803 15 ml

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, which is stored at –20°C (packaged separately).

Species Cross-Reactivity

H

Reactivity Key:  H=Human
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

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). 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

ELISA - Western correlation

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.

Sensitivity

Sensitivity

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.

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 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).

  1. Martin, F.H. et al. (1990) Cell 63, 203-11.
  2. Yarden, Y. et al. (1987) EMBO J 6, 3341-51.
  3. Gommerman, J.L. et al. (1997) J Biol Chem 272, 30519-25.
  4. Sattler, M. et al. (1997) J Biol Chem 272, 10248-53.
  5. Nocka, K. et al. (1990) EMBO J 9, 1805-13.
  6. Hirota, S. et al. (1998) Science 279, 577-80.
  7. Blume-Jensen, P. et al. (2000) Nat Genet 24, 157-62.

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