Product Pathways - HTScan Kinase Assay Kits
HTScan® FGF Receptor 3 Kinase Assay Kit #7734
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 |
| FLT3 (Tyr589) Biotinylated Peptide # 1305 | 1.25 milliliters |
| FGF Receptor 3 Kinase # 7733 | 5 micrograms |
Description
The kit provides a means of performing kinase activity assays with recombinant human FGFR-3 kinase. It includes active FGFR-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-FLT3 (Tyr589): 1,945Daltons. GST-FGFR-3 Kinase: 82 kDa.
Peptide Core Sequence
NEY*FY*V
Kinase Assay - Radiometric
Figure 1. FGFR-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: Poly(EY) 0.5 µg/50 µl, recombinant FGFR-3: 100 ng/50 µl.
Kinase Assay - DELFIA
Figure 3. Dose dependence curve of FGFR-3 kinase activity: DELFIA® data generated using Phospho-Tyrosine mAb (P-Tyr-100) #9411 to detect phosphorylation of substrate peptide (#1305) by FGFR-3 kinase. In a 50 µl reaction, increasing amounts of FGFR-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 FGFR-3 kinase activity: DELFIA® data generated using Phospho-Tyrosine mAb (P-Tyr-100) #9411 to detect phosphorylation of FGFR-3 substrate peptide (#1305) by FGFR-3 kinase. In a 50 µl reaction, 50 ng FGFR-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 FGFR-3 kinase activity: DELFIA® data generated using Phospho-Tyrosine mAb (P-Tyr-100) #9411 to detect phosphorylation of substrate peptide (#1305) by FGFR-3 kinase. In a 50 µl reaction, 50 ng of FGFR-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 FGFR-3 kinase activity: DELFIA® data generated using Phospho-Tyrosine mAb (P-Tyr-100) #9411 to detect phosphorylation of FGFR-3 substrate peptide (#1305) by FGFR-3 kinase. In a 50 µl reaction, 50 ng FGFR-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 FGFR-3 (Glu322-Thr806) (GenBank Accession No. NM_000142) 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 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 FGFR-3 kinase was quality controlled for purity by SDS-PAGE followed by Coomassie stain and Western blot. The specific activity of the FGFR-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 FGFR-3 activity using the FGFR-3 substrate peptide provided in this kit. FGFR-3 sensitivity to the inhibitor staurosporine was measured using the FGFR-3 substrate peptide provided in this kit [Fig.5].
Background
Fibroblast growth factors (FGFs) produce mitogenic and angiogenic effects in target cells by signaling through cell surface receptor tyrosine kinases. There are four members of the FGF receptor family: FGFR-1 (flg), FGFR-2 (bek, KGFR), FGFR-3 and FGFR-4. Each receptor contains an extracellular ligand binding domain, a transmembrane domain and a cytoplasmic kinase domain (1). Following ligand binding and dimerization, the receptors are phosphorylated at specific tyrosine residues (2). Seven tyrosine residues in the cytoplasmic tail of FGFR-1 can be phosphorylated: Tyr463, Tyr583, Tyr585, Tyr653, Tyr654, Tyr730 and Tyr766. Tyrosines 653 and 654 are important for catalytic activity of activated FGFR and are essential for signaling (3). The other phosphorylated tyrosine residues may provide docking sites for downstream signaling components such as Crk and PLCγ (4,5).
- Powers, C.J. et al. (2000) Endocr Relat Cancer 7, 165-97.
- Reilly, J.F. et al. (2000) J Biol Chem 275, 7771-8.
- Mohammadi, M. et al. (1996) Mol Cell Biol 16, 977-89.
- Mohammadi, M. et al. (1991) Mol Cell Biol 11, 5068-78.
- Larsson, H. et al. (1999) J Biol Chem 274, 25726-34.
Application References
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