Product Pathways - Jak/Stat Pathway
Jak2 Kinase #7751
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 murine Jak2 kinase (Lys752-Val1129), supplied as a GST fusion protein.
Source / Purification
The GST-Jak2 fusion protein was produced using a baculovirus expression system with a construct expressing murine Jak2 (Lys752-Val1129) (GenBank accession No. L16956) 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-Jak2 fusion protein was analyzed using SDS/PAGE followed by anti-Jak2 Western blot (A) or Silver stain (B).
Kinase Assay - Radiometric
Figure 2. Jak2 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: PolyEY, 1.5 µg/50 µl and 50 ng/50 µl Recombinant Jak2.
Kinase Assay - DELFIA
Figure 3. Dose dependence curve of Jak2 kinase activity: DELFIA® data generated using Phospho-Tyrosine mAb #9411 to detect phosphorylation of substrate peptide (#1305) by Jak2 kinase. In a 50 µl reaction, increasing amounts of Jak2 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-Jak2 fusion protein is 71 kDa. The purified kinase was quality controlled for purity using SDS-PAGE followed by Silver stain and Western blot [Fig.1]. Jak2 kinase activity was determined using a radiometric assay [Fig.2]. A kinase dose dependency assay was performed to measure Jak2 activity using HTScan™ Jak2 Kinase Assay Kit #7752 [Fig.3].
Background
Members of the Janus family of tyrosine kinases (Jak1, Jak2, Jak3 and Tyk2) are activated by ligands binding to a number of associated cytokine receptors (1). Upon cytokine receptor activation, Jak proteins become autophosphorylated and phosphorylate their associated receptors to provide multiple binding sites for signaling proteins. These associated signaling proteins, such as Stats (2), Shc (3), insulin receptor substrates (4) and focal adhesion kinase (FAK) (5), typically contain SH2 or other phospho-tyrosine-binding domains.
- Leonard, W.J. and O'Shea, J.J. (1998) Annu. Rev. Immunol. 16, 293-322.
- Darnell, J.E. (1997) Science 277, 1630-1635.
- VanderKuur, J. et al. (1995) J. Biol. Chem. 270, 7587-7593.
- Argetsinger, L.S. et al. (1995) J. Biol. Chem. 270, 14685-14692.
- Zhu, T. et al. (1998) J. Biol. Chem. 273, 10682-10689.
Application References
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