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7788
HTScan® VEGF Receptor 2 Kinase Assay Kit
Cellular Assay Kits

HTScan® VEGF Receptor 2 Kinase Assay Kit #7788

This product is discontinued

Kinase Assay - Radiometric Image 1

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.

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Kinase Assay - DELFIA Image 2

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.)

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Kinase Assay - DELFIA Image 3

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.)

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Kinase Assay - DELFIA Image 4

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.)

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Kinase Assay - DELFIA Image 5

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.)

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Product Includes Quantity (with Count) Solution Color
DTT (1000X) 1 x 80 µl
ATP (10 mM) 9804 1 x 1 ml

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.

Peptide Core Sequence:

EAY*GW

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.
Entrez-Gene Id
3791
Swiss-Prot Acc.
P35968
For Research Use Only. Not For Use In Diagnostic Procedures.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
DELFIA is a registered trademark of PerkinElmer, Inc.

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