Product Pathways - HTScan Kinase Assay Kits
HTScan® Syk Kinase Assay Kit #7779
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 |
| Gastrin Precursor (Tyr87) Biotinylated Peptide # 1310 | 1.25 milliliters |
| Syk Kinase # 7368 | 5 micrograms |
Description
The kit provides a means of performing kinase activity assays with recombinant human Syk kinase. It includes active Syk 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
Peptide substrate, Biotin-Gastrin Precursor (Tyr87): 2,853 Daltons. GST-Syk Kinase: 104 kDa
Peptide Core Sequence
EAY*GW
Kinase Assay - Radiometric
Figure 1. Syk kinase activity was measured in a radiometric assay using the following reaction conditions: 5 mM MOPS, pH 7.2, 2.5 mM β-glycerophosphate, 1 mM EGTA, 0.4 mM EDTA, 4 mM MgCl2, 2.5 mM MnCl2, 0.05 mM DTT, 50 μM ATP, Substrate: Poly EY, 400 ng/μL, and recombinant Syk: variable.
Kinase Assay - DELFIA
Figure 3. Dose dependence curve of Syk kinase activity: DELFIA® data generated using Phospho-Tyrosine Mouse mAb (P-Tyr-100) #9411 to detect phosphorylation of substrate peptide (#1310) by Syk kinase. In a 50 µl reaction, increasing amounts of Syk 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 Syk kinase activity: DELFIA® data generated using Phospho-Tyrosine Mouse mAb (P-Tyr-100) #9411 to detect phosphorylation of Syk substrate peptide (#1310) by Syk kinase. In a 50 µl reaction, 50 ng Syk, 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 Syk kinase activity: DELFIA® data generated using Phospho-Tyrosine Mouse mAb (P-Tyr-100) #9411 to detect phosphorylation of substrate peptide (#1310) by Syk kinase. In a 50 µl reaction, 50 ng of Syk 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 Syk kinase activity: DELFIA® data generated using Phospho-Tyrosine Mouse mAb (P-Tyr-100) #9411 to detect phosphorylation of Syk substrate peptide (#1310) by Syk kinase. In a 50 µl reaction, 50 ng Syk 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 full length human Syk (Met1-Asn635) (GenBank Accession No. NM_ 003177) 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 Mouse 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 Syk kinase was quality controlled for purity by SDS-PAGE followed by Coomassie stain. Syk 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 Syk activity using the Syk substrate peptide provided in this kit. Syk sensitivity to the inhibitor staurosporine was measured using the Syk substrate peptide provided in this kit [Fig.5].
Background
Syk is a protein tyrosine kinase that plays an important role in intracellular signal transduction in hematopoietic cells (1-3). Syk interacts with immunoreceptor tyrosine-based activation motifs (ITAMs) located in the cytoplasmic domains of immune receptors (4). It couples the activated immunoreceptors to downstream signaling events that mediate diverse cellular responses, including proliferation, differentiation and phagocytosis (4). There is also evidence of a role for Syk in nonimmune cells, and Syk is a potential tumor suppressor in human breast carcinomas (5). Tyr323 is a negative regulatory phosphorylation site within the SH2-kinase linker region in Syk. Phosphorylation of Tyr323 provides a direct binding site to the TKB domain of Cbl (6,7). Tyrosine 352 of Syk is involved in the association of PLC-γ1 (8). Tyrosines 525 and 526 are located in the activation loop of the Syk kinase domain, and phosphorylation of Tyr525/526 of human Syk (equivalent to the Tyr519/520 of mouse Syk) is essential for Syk function (9).
- Cheng, A.M. and Chan, A.C. (1997) Curr. Opin. Immunol. 9, 528-533.
- Kurosaki, T. et al. (1997) Curr. Opin. Immunol. 9, 309-318.
- Chu, D.H. et al. (1998) Immunol. Rev. 165, 167-180.
- Turner, M. et al. (2000) Immunol. Today 21, 148-154.
- Coopman, P.J. et al. (2000) Nature 406, 742-747.
- Decker, M. et al. (1998) J. Biol. Chem. 273, 8867-8874.
- Rao, N. et al. (2001) EMBO J. 20, 7085-7095.
- Law, C.L. et al. (1996) Mol. Cell. Biol. 16, 1305-1315.
- Zhang, J. et al. (2000) J. Biol. Chem. 275, 35442-35447.
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!
Companion Products
- 7450 Tyrosine Kinase Substrate Screening Kit
- 7368 Syk Kinase
- 9411 Phospho-Tyrosine Mouse mAb (P-Tyr-100)
- 1310 Gastrin Precursor (Tyr87) Biotinylated Peptide
- 9953 Staurosporine
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.
