Product Pathways - HTScan Kinase Assay Kits
HTScan® VEGF Receptor 2 Kinase Assay Kit #7788
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 |
| VEGF Receptor 2 Kinase # 7407 | 2 x 5 micrograms |
Description
The kit provides a means of performing kinase activity assays with recombinant human VEGFR-2 kinase. It includes active VEGFR-2 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) peptide: 2,853Daltons. GST-VEGFR-2 Kinase: 110 kDa.
Peptide Core Sequence
EAY*GW
Kinase Assay - Radiometric
Figure 1. VEGFR2 kinase activity was measured in a radiometric assay using the following reaction conditions: 4 mM MOPS, pH 7.2, 2.5 mM β-glycerophosphate, 1 mM EGTA, 0.4 mM EDTA, 4 mM MgCl2, 0.05 mM DTT, 50 μM ATP, Substrate: MBP 200 ng/μL, and variable amounts of recombinant VEGFR-2.
Kinase Assay - DELFIA
Figure 3. Dose dependence curve of VEGFR-2 kinase activity: DELFIA® data generated using Phospho-Tyrosine Mouse mAb (P-Tyr-100) #9411 to detect phosphorylation of substrate peptide #1310 by VEGFR-2 kinase. In a 50 µl reaction, increasing amounts of VEGFR-2 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 VEGFR-2 kinase activity: DELFIA® data generated using Phospho-Tyrosine Mouse mAb (P-Tyr-100) #9411 to detect phosphorylation of VEGFR-2 substrate peptide (#1310) by VEGFR-2 kinase. In a 50 µl reaction, 100 ng VEGFR-2, 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 VEGFR-2 kinase activity: DELFIA® data generated using Phospho-Tyrosine Mouse mAb (P-Tyr-100) #9411 to detect phosphorylation of substrate peptide #1310 by VEGFR-2 kinase. In a 50 µl reaction, 100 ng of VEGFR-2 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 VEGFR-2 kinase activity: DELFIA® data generated using Phospho-Tyrosine Mouse mAb (P-Tyr-100) #9411 to detect phosphorylation of VEGFR-2 substrate peptide #1310 by VEGFR-2 kinase. In a 50 µl reaction, 100 ng VEGFR-2 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 a fragment of human VEGFR-2 (Val789-Val1356) (GenBank accession No. NM_002253) 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 Substrates 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 VEGFR-2 kinase was quality controlled for purity by SDS-PAGE followed by Coomassie stain. The specific activity of the VEGFR-2 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 VEGFR-2 activity using the VEGFR-2 substrate peptide provided in this kit. VEGFR-2 sensitivity to the inhibitor staurosporine was measured using the VEGFR-2 substrate peptide provided in this kit [Fig.5].
Background
Vascular endothelial growth factor receptor 2 (VEGFR2, KDR, Flk-1) is a major receptor transducing VEGF-induced signaling in endothelial cells. Upon ligand binding, VEGFR2 undergoes autophosphorylation and becomes activated (1). Major autophosphorylation sites of VEGFR2 are located in the kinase insert domain (Tyr951/996) and in the tyrosine kinase catalytic domain (Tyr1054/1059) (2). Activation of the receptor leads to rapid recruitment of adaptor proteins, including Shc, GRB2, PI-3 kinase, Nck and the protein tyrosine phosphatases SHP-1 and SHP-2 (3). The phosphorylation of Tyr1212 provides a docking site for Grb2 binding and phospho-Tyr1175 binds with the p85 subunit of PI-3 kinase and PLCγ, as well as Shb (1,4,5). Signaling from VEGFR2 is necessary for the execution of VEGF-stimulated proliferation, chemotaxis and sprouting, as well as survival of cultured endothelial cells in vitro and angiogenesis in vivo (6-8).
- Meyer, M. et al. (1999) EMBO J. 18, 363-374.
- Dougher-Vermazen, M. et al. (1994) Biochem. Biophys. Res. Commun. 205, 728-738.
- Kroll, J. and Waltenberger, J. (1997) J. Biol. Chem. 272, 32521-32527.
- Takahashi, T. et al. (2001) EMBO J. 20, 2768-2778.
- Holmqvist, K. et al. (2004) J. Biol. Chem. 279, 22267-22275.
- Karkkainen, M.J. and Petrova, T. (2000) Oncogene 19, 5598-5605.
- Rahimi, N. et al. (2000) J. Biol. Chem. 275, 16986-16992.
- Claesson-Welsh, L. (2003) Biochem. Soc. Trans. 31, 20-24.
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
- 7407 VEGF Receptor 2 Kinase
- 9411 Phospho-Tyrosine Mouse mAb (P-Tyr-100)
- 1310 Gastrin Precursor (Tyr87) Biotinylated Peptide
- 9953 Staurosporine
- 9805 HTScan® Tyrosine Kinase Buffer (4X)
- 9804 ATP (10 mM)
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.
