Product Pathways - HTScan Kinase Assay Kits
HTScan® c-Kit Kinase Assay Kit #7755
Cell Signaling Technology offers a full line of protein kinases, substrates, antibody detection reagents and HTScan® kits. Browse our "Reagents for High-Throughput Screening" product listing or contact us at drugdiscovery@cellsignal.com.
| Kit Includes | Quantity |
|---|---|
| Phospho-Tyrosine Mouse mAb (P-Tyr-100) # 9411 | 30 microliters |
| HTScan® Tyrosine Kinase Buffer (4X) # 9805 | 15 milliliters |
| DTT (1000X, 1.25 M) | 80 microliters |
| ATP (10 mM) # 9804 | 1 milliliters |
| c-Kit Kinase # 7754 | 5 micrograms |
| KDR (Tyr996) Biotinylated Peptide # 1364 | 1.25 milliliters |
Description
The kit provides a means of performing kinase activity assays with recombinant human c-Kit kinase. It includes active c-Kit kinase (supplied as a GST fusion protein), a biotinylated peptide substrate and a phospho-tyrosine antibody for detection of the phosphorylated form of the substrate peptide.
Molecular Weights
Peptide substrate, Biotin-KDR (Tyr996): 2,164 Daltons, GST-c-Kit Kinase: 75 KDa.
Peptide Core Sequence
DLY*KD
Kinase Assay - Radiometric
Figure 1. c-Kit kinase activity was measured in a radiometric assay using the following reaction conditions: 60 mM HEPES-NaOH, pH 7.5, 3 mM MgCl2, 3 mM MnCl2, 3 µM Na-orthovanadate, 1.2 mM DTT, ATP (variable), 2.5 µg/50 µl PEG20.000, Substrate: Tetra (LRRWSLG), 5 µg/50 µl, recombinant c-Kit: 200 ng/50 µl.
Kinase Assay - DELFIA
Figure 3. Dose dependence curve of c-Kit kinase activity: DELFIA® data generated using Phospho-Tyrosine mAb (P-Tyr-100) #9411 to detect phosphorylation of substrate peptide (#1364) by c-Kit kinase. In a 50 µl reaction, increasing amounts of c-Kit and 1.5 µM substrate peptide were used per reaction at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Kinase Assay - DELFIA
Figure 5. Staurosporine inhibition of c-Kit kinase activity: DELFIA® data generated using Phospho-Tyrosine mAb (P-Tyr-100) #9411 to detect phosphorylation of c-Kit substrate peptide (#1364) by c-Kit kinase. In a 50 µl reaction, 100 ng c-Kit, 1.5 µM substrate peptide, 20 µM ATP and increasing amounts of staurosporine were used per reaction at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Kinase Assay - DELFIA
Figure 4. Peptide concentration dependence of c-Kit kinase activity: DELFIA® data generated using Phospho-Tyrosine mAb (P-Tyr-100) #9411 to detect phosphorylation of substrate peptide (#1364) by c-Kit kinase. In a 50 µl reaction, 100 ng of c-Kit and increasing concentrations of substrate peptide were used per reaction at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Kinase Assay - DELFIA
Figure 2. Time course of c-Kit kinase activity: DELFIA® data generated using Phospho-Tyrosine mAb (P-Tyr-100) #9411 to detect phosphorylation of KDR substrate peptide (#1364) by c-Kit kinase. In a 50 µl reaction, 100 ng c-Kit and 1.5 µM substrate peptide were used per reaction. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Source / Purification
The GST-c-Kit fusion protein was produced using a baculovirus expression system with a construct expressing a fragment of human c-Kit (Thr544-Val976) with an amino-terminal GST tag. The protein was purified by one-step affinity chromatography using glutathione-agarose.
Quality Control
The substrate peptide was selected using our Tyrosine Kinase Substrate Screening c-Kit #7450. Phospho-Tyrosine mAb (P-Tyr-100) #7450 was used for detection. The quality of the biotinylated peptide was evaluated by reverse-phase HPLC and by mass spectrometry.Purified c-Kit kinase was quality controlled for purity by SDS-PAGE followed by silver stain and Western blot. The specific activity of the c-Kit kinase was determined using a radiometric assay [Fig.1]. Time course [Fig.2], kinase dose-dependency [Fig.3] and substrate dose-dependency [Fig.4] assays were performed to verify c-Kit activity using the c-Kit substrate peptide provided in this kit. c-Kit sensitivity to the inhibitor staurosporine was measured using the c-Kit substrate peptide provided in this kit [Fig.5].
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), 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, 203-11.
- 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.
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!
Companion Products
- 7450 Tyrosine Kinase Substrate Screening Kit
- 7754 c-Kit Kinase
- 9411 Phospho-Tyrosine Mouse mAb (P-Tyr-100)
- 1364 KDR (Tyr996) Biotinylated Peptide
- 9953 Staurosporine
- 9805 HTScan® Tyrosine Kinase Buffer (4X)
- 9804 ATP (10 mM)
This product is for in vitro research use only and is not intended for use in humans or animals. This product is not intended for use as therapeutic or in diagnostic procedures.
