Product Pathways - HTScan Kinase Assay Kits
HTScan® Jak2 Kinase Assay Kit #7752
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 |
| DTT (1000x, 1.25 M) | 80 microliters |
| ATP (10 mM) # 9804 | 1 milliliters |
| Jak2 Kinase # 7751 | 2 x 5 micrograms |
| FLT3 (Tyr589) Biotinylated Peptide # 1305 | 1.25 milliliters |
Description
The kit provides a means of performing kinase activity assays with recombinant murine Jak2 kinase. It includes active Jak2 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.
Molecular Weights
FLT3 (Tyr589): 1,945 Daltons, GST-Jak2 Kinase: 71 kDa Daltons.
Peptide Core Sequence
NEY*FY*V
Kinase Assay - Radiometric
Figure 1. Jak2 kinase activity was measured in a radioisotopic filter binding 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 Jak2: 50 ng/50 µl.
Kinase Assay - DELFIA
Figure 3. Dose dependence curve of Jak2 kinase activity: DELFIA® data generated using Phospho-Tyrosine mAb (P-Tyr-100) #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.)
Kinase Assay - DELFIA
Figure 5. Staurosporine inhibition of Jak2 kinase activity: DELFIA® data generated using Phospho-Tyrosine mAb (P-Tyr-100) #9411 to detect phosphorylation of Jak2 substrate peptide (#1305) by Jak2 kinase. In a 50 µl reaction, 100 ng Jak2, 1.5 µM substrate peptide, 20 µM ATP and increasing amounts of staurosporine were used per reaction at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Kinase Assay - DELFIA
Figure 4. Peptide concentration dependence of Jak2 kinase activity: DELFIA® data generated using Phospho-Tyrosine mAb (P-Tyr-100) to detect phosphorylation of substrate peptide (#1305) by Jak2 kinase. In a 50 µl reaction, 100 ng of Jak2 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 Jak2 kinase activity: DELFIA® data generated using Phospho-Tyrosine mAb (P-Tyr-100) to detect phosphorylation of Jak2 substrate peptide (#1305) by Jak2 kinase. In a 50 µl reaction, 100 ng Jak2 and 1.5 µM substrate peptide were used per reaction. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Source / Purification
The GST-Jak2 Kinase fusion protein was produced using a baculovirus expression system with a construct expressing a fragment of 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.
Quality Control
The substrate peptide was selected using our Tyrosine Kinase Substrate Screening Kit #7450. Phospho-Tyrosine mAb (P-Tyr-100) #9411 was used for detection. The quality of the biotinylated peptide was evaluated by reverse-phase HPLC and by mass spectrometry.Purified Jak2 kinase was quality controlled for purity by SDS-PAGE followed by silver stain and Western blot. The specific activity of the Jak2 kinase was determined using a radiometric filter binding assay [Fig.1]. Time course [Fig.2], kinase dose-dependency [Fig.3] and substrate dose-dependency [Fig.4] assays were performed to verify Jak2 activity using the Jak2 substrate peptide provided in this kit. Jak2 sensitivity to the inhibitor staurosporine was measured using the Jak2 substrate peptide provided in this kit [Fig.5].
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
Have you published research involving the use of our products? If so we'd love to hear about it. Please let us know!
