Revision 1
Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
:

:

:

:

UniProt ID:

#P35968

Entrez-Gene Id:

3791

Product Information

Storage

Antibodies are supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol. Do not aliquot the antibodies. Peptides are supplied at 6 µM in 0.001% DMSO. Enzymes are supplied in 50 mM Tris-HCL (pH 8.0), 100 mM NaCl,
5 mM DTT, 15 mM reduced glutathione and 20%
glycerol. Store at –80°C.
Keep enzymes on ice during use.
Avoid repeated freeze-thaw cycles.

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.

Product 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 Formula Peptide substrate, Biotin-Gastrin Precursor (Tyr87) peptide: 2,853Daltons. GST-VEGFR-2 Kinase: 110 kDa.

Background

Vascular endothelial growth factor receptor 2 (VEGFR2, KDR, Flk-1) is a major receptor for 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, PI3 kinase, NCK, and the protein tyrosine phosphatases SHP-1 and SHP-2 (3). Phosphorylation at Tyr1212 provides a docking site for GRB2 binding and phospho-Tyr1175 binds the p85 subunit of PI3 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).

  1. Meyer, M. et al. (1999) EMBO J 18, 363-74.
  2. Dougher-Vermazen, M. et al. (1994) Biochem Biophys Res Commun 205, 728-38.
  3. Kroll, J. and Waltenberger, J. (1997) J Biol Chem 272, 32521-7.
  4. Takahashi, T. et al. (2001) EMBO J 20, 2768-78.
  5. Holmqvist, K. et al. (2004) J Biol Chem 279, 22267-75.
  6. Karkkainen, M.J. and Petrova, T.V. (2000) Oncogene 19, 5598-605.
  7. Rahimi, N. et al. (2000) J Biol Chem 275, 16986-92.
  8. Claesson-Welsh, L. (2003) Biochem Soc Trans 31, 20-4.

Species Reactivity

Species reactivity is determined by testing in at least one approved application (e.g., western blot).

Cross-Reactivity Key

H: human M: mouse R: rat Hm: hamster Mk: monkey Vir: virus Mi: mink C: chicken Dm: D. melanogaster X: Xenopus Z: zebrafish B: bovine Dg: dog Pg: pig Sc: S. cerevisiae Ce: C. elegans Hr: horse GP: Guinea Pig Rab: rabbit All: all species expected

Trademarks and Patents

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

Limited Uses

Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.

Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

Orders: 877-616-CELL (2355)•[email protected]• Support: 877-678-TECH (8324)•[email protected]• Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.
Revision 1
#7788

HTScan® VEGF Receptor 2 Kinase Assay Kit

HTScan® VEGF Receptor 2 Kinase Assay Kit: Image 1 Expand Image
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.)
HTScan® VEGF Receptor 2 Kinase Assay Kit: Image 2 Expand Image
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.)
HTScan® VEGF Receptor 2 Kinase Assay Kit: Image 3 Expand Image
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.)
HTScan® VEGF Receptor 2 Kinase Assay Kit: Image 4 Expand Image
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.)
HTScan® VEGF Receptor 2 Kinase Assay Kit: Image 5 Expand Image
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.
Image Gallery
Learn more about how we get our images
Orders: 877-616-CELL (2355)•[email protected]• Support: 877-678-TECH (8324)•[email protected]• Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.