Product Pathways - Tyrosine Kinase/ Adaptors
VEGF Receptor 3 Kinase #7790
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.
Description
Purified recombinant human VEGFR-3 kinase (Asn799-Arg1298), supplied as a GST fusion protein.
Source / Purification
The GST-Kinase fusion protein was produced using a baculovirus expression system with a construct expressing human VEGFR-3 (Asn799-Arg1298) (GenBank accession No. NM_002020) with an amino-terminal GST tag. The protein was purified by one-step affinity chromatography using glutathione-agarose.
Gel Staining
Figure 1. The purity of the GST-VEGFR-3 fusion protein was analyzed using SDS/PAGE followed by anti-VEGFR-3 Western blot (A) or Silver stain (B).
Kinase Assay - Radiometric
Figure 2. VEGFR-3 kinase activity was measured in a radiometric assay using the following reaction conditions: 60 mM HEPES-NaOH, pH 7.5, 3 mM MgCl2, 3 mM MnCl2, 3 µM Na-orthovanadate, 1.2 mM DTT, ATP (variable), 2.5 µg/50 µl PEG20,000, Substrate: PolyEY, 5 µg/50 µl and 100 ng/50 µl Recombinant VEGFR-3.
Kinase Assay - DELFIA
Figure 3. Dose dependence curve of VEGFR-3 kinase activity: DELFIA® data generated using Phospho-Tyrosine Mouse mAb (P-Tyr-100) #9411 to detect phosphorylation of substrate peptide (#1367) by VEGFR-3 kinase. In a 50 µl reaction, increasing amounts of VEGFR-3 and 1.5 µM substrate peptide were used per reaction at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Quality Control
The theoretical molecular weight of the GST-VEGFR-3 fusion protein is 86 kDa. The purified kinase was quality controlled for purity using SDS-PAGE followed by Silver stain and Western blot [Fig.1]. VEGFR-3 kinase activity was determined using a radiometric assay [Fig.2]. A kinase dose dependency assay was performed to measure VEGFR-3 activity using HTScan™ VEGFR-3 Kinase Assay Kit #7791 [Fig.3].
Background
Vascular endothelial growth factor receptor 3 (VEGFR3) is a 195 kDa membrane receptor tyrosine kinase. VEGF receptors are characterized by the presence of seven extracellular immunoglobulin (Ig)-like domains followed by a membrane-spanning domain and a conserved intracellular tyrosine kinase domain (1). VEGF Receptor 3 expression is largely restricted to adult lymphatic endothelium and is thought to control lymphangiogenesis (1,2). Binding of VEGF-C/VEGF-D to VEGFR3 results in transphosphorylation of tyrosine residues in its intracellular domain, recruitment of signaling molecules and activation of ERK1/2 and Akt signaling cascades (1,3).
- Robinson, C.J. and Stringer, S.E. (2001) J. Cell Sci.114, 853-865.
- Valtola, R. et al. (1999) Am. J. Pathol. 154, 1381-1390.
- Saharinen, P. and Petrova, T.V. (2004) Ann. N.Y. Acad. Sci. 1014, 76-87.
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
- 7791 HTScan® VEGF Receptor 3 Kinase Assay Kit
- 1367 Met (Tyr1235) Biotinylated Peptide
- 9411 Phospho-Tyrosine Mouse mAb (P-Tyr-100)
- 9805 HTScan® Tyrosine Kinase Buffer (4X)
- 9804 ATP (10 mM)
- 9953 Staurosporine
- 7450 Tyrosine Kinase Substrate Screening Kit
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.
