Product Pathways - HTScan Kinase Assay Kits
HTScan® Csk Kinase Assay Kit #7802
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 |
| Pyk2 (Tyr881) Biotinylated Peptide # 1370 | 1.25 milliliters |
| Csk Kinase # 7801 | 5 micrograms |
Description
The kit provides a means of performing kinase activity assays with recombinant human Csk kinase. It includes active Csk 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-peptide: 2,290 Daltons, GST-Csk Kinase: 80 kDa.
Peptide Core Sequence
LVY*LN
Kinase Assay - Radiometric
Figure 1. CSK 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 uM Na-orthovanadate, 1.2 mM DTT, ATP (variable), 200 ng/50 µl PEG20,000, Substrate: Poly(EY) 1 ug/50 µl, recombinant Csk: 4 Units/50 µl.
Kinase Assay - Radiometric
Figure 3. Dose dependence curve of Csk kinase activity: DELFIA® data generated using Phospho-Tyrosine mAb (P-Tyr-100) #9411 to detect phosphorylation of substrate peptide (#1370) by Csk kinase. In a 50 µl reaction, increasing amounts of Csk 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 - Radiometric
Figure 5. Staurosporine inhibition of Csk kinase activity: DELFIA® data generated using Phospho-Tyrosine mAb (P-Tyr-100) #9411 to detect phosphorylation of Csk substrate peptide (#1370) by Csk kinase. In a 50 µl reaction, 100 ng Csk kinase, 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 - Radiometric
Figure 4. Peptide concentration dependence of Csk kinase activity: DELFIA® data generated using Phospho-Tyrosine mAb (P-Tyr-100) #9411 to detect phosphorylation of substrate peptide (#1370) by Csk kinase. In a 50 µl reaction, 100 ng of Csk 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 - Radiometric
Figure 2. Time course of Csk kinase activity: DELFIA® data generated using Phospho-Tyrosine mAb (P-Tyr-100) #9411 to detect phosphorylation of Csk substrate peptide (#1370) by Csk kinase. In a 50 µl reaction, 100 ng Csk 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 Met1-Leu450 of the human Csk (GenBank Accession No.NM_004383) with an amino-terminal GST tag. The protein was then 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 Csk kinase was quality controlled for purity by SDS-PAGE followed by Coomassie stain and Western blot. The specific activity of the Csk kinase 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 Csk activity using the Csk substrate peptide provided in this kit. Csk sensitivity to the inhibitor staurosporine was measured using the Csk substrate peptide provided in this kit [Fig.5].
Background
Carboxy-terminal Src kinase (Csk) is a ubiquitously expressed nonreceptor tyrosine kinase that negatively regulates the Src family kinases (SFKs) by phosphorylation of the SFK carboxy-terminal tyrosine (1,2). Phosphorylated carboxy-terminal tyrosine binds to the SH2 domain of SFK intramolecularly and leads to folding and inactivation of the SFK (3). This Csk-catalyzed SFK tyrosine phosphorylation is highly specific and exclusive. The SFK carboxy-terminal tyrosine is the only known physiological substrate of Csk (4).Csk consists of an SH2, an SH3, and a kinase domain. There is evidence that the SH2 and SH3 domains are essential for the regulation of SFK, and Csk can be recruited to the membrane where SFKs are in an active state. This process is mediated by a Csk-binding protein (Cbp, also called PAG), which binds tightly to the SH2 domain of Csk (5). Activation of SFK by extracellular stimuli leads to the tyrosine phosphorylation of Cbp, generating docking sites for Csk. The recruitment of Csk forms a feedback mechanism for termination of SFK function (6).
- Nada, S. et al. (1991) Nature 351, 69-72.
- Nada, S. et al. (1993) Cell 73, 1125-1135.
- Lee, S. et al. (2003) Proc. Natl. Acad. Sci. USA 100, 14707-14712.
- Imamoto, A. and Soriano, P. (1993) Cell 73, 1117-1124.
- Kawabuchi, M. et al. (2000) Nature 404, 999-1003.
- Matsuoka, H. et al. (2004) J. Biol. Chem. 279, 5975-5983.
Application References
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