Figure 3. Dose dependence curve of Abl1 T315I kinase activity: DELFIA® data generated using Phospho-Tyrosine Mouse mAb (P-Tyr-100) #9411 to detect phosphorylation of substrate peptide (#1366) by Abl1 T315I kinase. In a 50 µl reaction, increasing amounts of Abl1 T315I and 1.5 µM substrate peptide were used per reaction at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Figure 5. Sensitivities of wild type and mutant Abl1 T315I kinase to Staurosporine (A) and Gleevec (B) were compared: DELFIA® data generated using Phospho-Tyrosine mAb (P-Tyr-100) #9411 to detect phosphorylation of Abl1 substrate peptide (#1366) by the Abl1 kinases. In a 50 µl reaction, 50 ng of kinase, 1.5 µM substrate peptide, 5 µM ATP and increasing concentration of indicated inhibitors were used per reaction well at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Figure 4. Peptide concentration dependence of Abl1 T315I kinase activity: DELFIA® data generated using Phospho-Tyrosine Mouse mAb (P-Tyr-100) #9411 to detect phosphorylation of substrate peptide (#1366) by Abl1 T315I kinase. In a 50 µl reaction, 50 ng of Abl1 T315I and increasing concentrations of substrate peptide were used per reaction at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Figure 2. Time course of Abl1 T315I kinase activity: DELFIA® data generated using Phospho-Tyrosine Mouse mAb (P-Tyr-100) #9411 to detect phosphorylation of Abl1 T315I substrate peptide (#1366) by Abl1 T315I kinase. In a 50 µl reaction, 50 ng Abl1 T315I and 1.5 µM substrate peptide were used per reaction. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Figure 1. Abl1 T315I kinase activity was measured in a radiometric assay using the following reaction conditions: 60 mM HEPES-NaOH, pH 7.5, 5 mM MgCl2, 5 mM MnCl2, 3 µM Na-orthovanadate, 1.2 mM DTT, 100 µM ATP, 100 µM Signal Transduction Protein (Tyr160) Biotinylated Peptide #1366 and variable amount of recombinant Abl1 T315I. Reaction mixture incubated at room temperature for 10 minutes.
The kit provides a means of performing kinase activity assays with recombinant human Abl1 T315I kinase. It includes active Abl1 T315I kinase (supplied as a GST fusion protein), a biotinylated peptide substrate and a phospho-tyrosine monoclonal antibody for detection of the phosphorylated form of the substrate peptide.
Peptide substrate, Biotin-Signal Transduction Protein (Tyr160): 1830 Daltons. GST-Abl1 T315I Kinase: 76 kDa.
The c-Abl proto-oncogene encodes a nonreceptor protein tyrosine kinase that is ubiquitously expressed and highly conserved in metazoan evolution. c-Abl protein is distributed in both the nucleus and the cytoplasm of cells. It is implicated in regulating cell proliferation, differentiation, apoptosis, cell adhesion, and stress responses (1-3). c-Abl kinase activity is increased in vivo by diverse physiological stimuli including integrin activation; PDGF stimulation; and binding to c-Jun, Nck, and RFX1 (2,4). The in vivo mechanism for regulation of c-Abl kinase activity is not completely understood. Tyr245 is located in the linker region between the SH2 and catalytic domains. This positioning is conserved among Abl family members. Phosphorylation at Tyr245 is involved in the activation of c-Abl kinase (5). In addition, phosphorylation at Tyr412, which is located in the kinase activation loop of c-Abl, is required for kinase activity (6).
Imatinib (Gleevec) has been successfully used to treat Bcr-Abl kinase associated chronic myeloid leukemia (CML). The most frequently identified mutation associated with resistance to Gleevec in CML patients is T315I in the Abl kinase domain (7,8).
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