Product Pathways - HTScan Kinase Assay Kits
HTScan® VEGF Receptor 1 Kinase Assay Kit #7785
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 1 Kinase # 7784 | 5 micrograms |
Description
The kit provides a means of performing kinase activity assays with recombinant human VEGFR-1 kinase. It includes active VEGFR-1 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 (Tlyr87): 2,853 Daltons. GST-VEGFR-1 Kinase: 89 kDa.
Peptide Core Sequence
EAY*GW
Kinase Assay - Radiometric
Figure 1. VEGFR-1 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-1: 100 ng/50 µl.
Kinase Assay - DELFIA
Figure 3. Dose dependence curve of VEGFR-1 kinase activity: DELFIA® data generated using Phospho-Tyrosine Mouse mAb (P-Tyr-100) #9411 to detect phosphorylation of substrate peptide (#1310) by VEGFR-1 kinase. In a 50 µl reaction, increasing amounts of VEGFR-1 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-1 kinase activity: DELFIA® data generated using Phospho-Tyrosine Mouse mAb (P-Tyr-100) #9411 to detect phosphorylation of VEGFR-1 substrate peptide (#1310) by VEGFR-1 kinase. In a 50 µl reaction, 50 ng VEGFR-1, 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-1 kinase activity: DELFIA® data generated using Phospho-Tyrosine Mouse mAb (P-Tyr-100) #9411 to detect phosphorylation of substrate peptide (#1310) by VEGFR-1 kinase. In a 50 µl reaction, 50 ng of VEGFR-1 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-1 kinase activity: DELFIA® data generated using Phospho-Tyrosine Mouse mAb (P-Tyr-100) #9411 to detect phosphorylation of VEGFR-1 substrate peptide (#1310) by VEGFR-1 kinase. In a 50 µl reaction, 50 ng VEGFR-1 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-1 (Lys784-Ile1338) 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-1 kinase was quality controlled for purity by SDS-PAGE followed by Coomassie stain and Western blot. The specific activity of the VEGFR-1 activity 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-1 activity using the VEGFR-1 substrate peptide provided in this kit. VEGFR-1 sensitivity to the inhibitor staurosporine was measured using the VEGFR-1 substrate peptide provided in this kit [Fig.5].
Background
The vascular endothelial growth factor (VEGF) receptor (VEGFR-1, Flt-1) is a 180 kDa receptor tyrosine kinase belonging to the VEGFR (Flt) family (1-3). The receptor is comprised of seven extracellular Ig-like domains, a single transmembrane region and cytoplasmic tail containing the active kinase domain (1,2). VEGFR-1 plays an important role in endothelial cell function and normal vascular development, as well as in hematopoietic function (2,3). VEGF-A is a high affinity ligand of VEGFR-1. VEGFR-1 also binds VEGF-B and PLGF (2). Ligand binding results in very little VEGFR-1 kinase activation, and VEGFR-1/VEGF-A binding negatively regulates VEGF function by diverting the growth factor from other functional VEGF receptors (3).
- Ferrara, N. et al. (2003) Nat Med 9, 669-76.
- Clauss, M. (2000) Semin Thromb Hemost 26, 561-9.
- Claesson-Welsh, L. (2003) Biochem Soc Trans 31, 20-4.
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
- 7784 VEGF Receptor 1 Kinase
- 9411 Phospho-Tyrosine Mouse mAb (P-Tyr-100)
- 1310 Gastrin Precursor (Tyr87) Biotinylated Peptide
- 9953 Staurosporine
- 9804 ATP (10 mM)
- 9805 HTScan® Tyrosine Kinase Buffer (4X)
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.
