Product Pathways - Kinases
Abl1 Kinase #7904
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.
Description
Purified recombinant of Abl1 kinase (Pro118-Ser553), supplied as a GST fusion protein.
Source / Purification
The GST-Abl1 Kinase fusion protein was produced using a baculovirus expression system with a construct expressing human Abl (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.
Gel Staining
Figure 1. The purity of the GST-Abl1 fusion protein was analyzed using SDS/PAGE followed by Coomassie stain.
Kinase Assay - Radiometric
Figure 2. Abl1 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 uM Signal Transduction Protein (Tyr160) Biotinylated Peptide (#1366) and variable amount of Recombinant Abl1. Reaction mixture incubated at room temperature for 10 minutes.
Kinase Assay - DELFIA
Figure 3. Dose dependence curve of Abl1 kinase activity: DELFIA® data generated using Phospho-Tyrosine mAb (P-Tyr-100) #9411 to detect phosphorylation of substrate peptide (#1366) by Abl1 kinase. In a 50 µl reaction, increasing amounts of Abl1 and 1.5 µM substrate peptide were used per reaction at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Quality Control
The theoretical molecular weight of the GST-Abl1 kinase fusion protein is 76 kDa. The purified kinase was quality controlled for purity using SDS-PAGE followed by Coomassie stain [Fig.1]. The specific activity of the Abl1 kinase was determined using a radiometric assay [Fig.2]. A kinase dose dependency assay was performed to measure Abl1 kinase activity using HTScan™ Abl1 Kinase Assay Kit #7905 [Fig.3].
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.
- 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.
Application References
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Companion Products
- 7905 HTScan® Abl1 Kinase Assay Kit
- 1366 Signal Transduction Protein (Tyr160) Biotinylated Peptide
- 9411 Phospho-Tyrosine Mouse mAb (P-Tyr-100)
- 9805 HTScan® Tyrosine Kinase Buffer (4X)
- 9804 ATP (10 mM)
- 9953 Staurosporine
- 7450 Tyrosine Kinase Substrate Screening Kit
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.
