Product Pathways - HTScan Kinase Assay Kits
HTScan® Abl1 T315I Kinase Assay Kit #7907
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 |
| Abl1 T315I Kinase # 7906 | 5 micrograms |
| ATP (10 mM) # 9804 | 1 milliliters |
| Signal Transduction Protein (Tyr160) Biotinylated Peptide # 1366 | 1.25 milliliters |
| DTT (1000x, 1.25M) | 80 microliters |
Description
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.
Molecular Weights
Peptide substrate, Biotin-Signal Transduction Protein (Tyr160): 1830 Daltons. GST-Abl1 T315I Kinase: 76 kDa.
Peptide Core Sequence
GIY*DV
Kinase Assay - Radiometric
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.
Kinase Assay - DELFIA
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.)
Kinase Assay - DELFIA
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.)
Kinase Assay - DELFIA
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.)
Kinase Assay - DELFIA
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.)
Source / Purification
The GST-kinase fusion protein was produced using a baculovirus expression system with a construct expressing human Abl1 T315I (Pro118-Ser553) (GenBank Accession No. NM_005157) 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 by screening Tyrosine Kinase Substrate Screening Kit #7450. Phospho-Tyrosine mAb (P-Tyr-100) #9411 was used for detection. The quality of the biotinylated peptides was evaluated by reverse-phase HPLC and by mass spectrometry.Purified Abl1 T315I kinase was quality controlled for purity by SDS-PAGE followed by Coomassie stain. Abl1 T315I kinase activity 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 Abl1 T315I activity using the Abl1 T315I substrate peptide provided in this kit. Abl1 T315I sensitivity to the inhibitor staurosporine and Gleevec was measured using the Abl1 T315I substrate peptide provided in this kit [Fig.5].
Background
The c-Abl proto-oncogene encodes a nonreceptor type 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 of 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 of Tyr245 is involved in the activation of c-Abl kinase (5). In addition, phosphorylation of Tyr412 which is located in the kinase activation loop of c-Abl is required for kinase activity (6). Thr735 is located within a conserved 14-3-3 protein binding motif region, and can be phosphorylated upon stress stimulation or TPA treatment (Wu, J. et al. unpublished data). Phosphorylation at Thr735 may play an important role in regulating c-Abl localization as well as its function.
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).
- Wang, J.Y. et al. (2000) Oncogene 19, 5643-5650.
- Van Etten, R.A. et al. (1999) Trends Cell. Biol. 9, 179-182.
- Danial, N.N. et al. (2000) Oncogene 19, 2523-2531.
- Shaul, Y. et al. (2000) Cell Death Differ. 7, 10-16.
- Brasher, B.B. et al. (2000) J. Biol. Chem. 275, 35631-35637.
- Pluk, H. et al. (2002) Cell 108, 247-259.
- Fabian, M.A. et al. (2005) Nat. Biotechnol. 23, 329-36.
- Carter, T.A. et al. (2005) Proc. Natl. Acad. Sci. USA 102, 11011-6.
Application References
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