Figure 1. 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 50ul reaction, increasing amounts of c-Kit and 1.5uM substrate peptide were used per reaction at room temperature for 30 minutes (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Figure 2. 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.
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.
Peptide substrate, Biotin-KDR (Tyr996): 2,164 Daltons, GST-c-Kit Kinase: 75 KDa.
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).
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