Product Pathways - Tyrosine Kinase/ Adaptors
VEGF Receptor 1 Kinase #7784
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 VEGF Receptor 1 kinase (Lys784-Ile1338), 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-1 (Lys784-Ile1338) (GenBank Accession No. NM_002019) 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-1 fusion protein was analyzed using SDS/PAGE followed by anti- VEGFR-1 Western blot (A) or Silver stain (B).
Kinase Assay - Radiometric
Figure 2. 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: PolyEY, 1.5 µg/50 µl and 100 ng/50 µl Recombinant VEGFR-1.
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.)
Quality Control
The theoretical molecular weight of the GST-VEGFR-1 fusion protein is 89.2 kDa. The purified kinase was quality controlled for purity using SDS-PAGE followed by Silver stain and Western blot [Fig.1]. VEGFR-1 kinase activity was determined using a radiometric assay [Fig.2]. A kinase dose dependency assay was performed to measure VEGFR-1 activity using HTScan™ VEGFR-1 Kinase Assay Kit #7785 [Fig.3].
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
- 7785 HTScan® VEGF Receptor 1 Kinase Assay Kit
- 1310 Gastrin Precursor (Tyr87) 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.
