Product Pathways - HTScan Kinase Assay Kits
HTScan® VEGF Receptor 3 Kinase Assay Kit #7791
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 |
| Met (Tyr1235) Biotinylated Peptide # 1367 | 1.25 milliliters |
| VEGF Receptor 3 Kinase # 7790 | 2 x 5 micrograms |
Description
The kit provides a means of performing kinase activity assays with recombinant human VEGFR-3 kinase. It includes active VEGFR-3 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-MET (Tyr1253) peptide: 2,200Daltons. GST-VEGFR-3 Kinase: 86 kDa.
Peptide Core Sequence
MY*DKEY*Y*S
Kinase Assay - Radiometric
Figure 1. 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: PolyEY5 µg/50 µl, recombinant VEGFR-3: 100 ng/50 µl.
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.)
Kinase Assay - DELFIA
Figure 5. Staurosporine inhibition of VEGFR-3 kinase activity: DELFIA® data generated using Phospho-Tyrosine Mouse mAb (P-Tyr-100) #9411 to detect phosphorylation of VEGFR-3 substrate peptide (#1367) by VEGFR-3 kinase. In a 50 µl reaction, 100 ng VEGFR-3, 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-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, 100 ng of VEGFR-3 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-3 kinase activity: DELFIA® data generated using Phospho-Tyrosine Mouse mAb (P-Tyr-100) #9411 to detect phosphorylation of VEGFR-3 substrate peptide (#1367) by VEGFR-3 kinase. In a 50 µl reaction, 100 ng VEGFR-3 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-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.
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 VEGFR-3 kinase was quality controlled for purity by SDS-PAGE followed by Coomassie stain and Western blot. The specific activity of the VEGFR-3 kinase 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-3 activity using the VEGFR-3 substrate peptide provided in this kit. VEGFR-3 sensitivity to the inhibitor staurosporine was measured using the VEGFR-3 substrate peptide provided in this kit [Fig.5].
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
- 7450 Tyrosine Kinase Substrate Screening Kit
- 7790 VEGF Receptor 3 Kinase
- 9411 Phospho-Tyrosine Mouse mAb (P-Tyr-100)
- 1367 Met (Tyr1235) 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.
