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HTScan® FGF Receptor 3 Kinase Assay Kit #7734
This product is discontinued
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.
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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.)
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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.)
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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.)
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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.)
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| Product Includes | Quantity (with Count) | Solution Color | |||
|---|---|---|---|---|---|
| DTT (1000X) | 1 x 80 µl | ||||
| ATP (10 mM) 9804 | 1 x 1 ml |
Product 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 Formula:Peptide substrate, Biotin-FLT3 (Tyr589): 1,945Daltons. GST-FGFR-3 Kinase: 82 kDa.
Peptide Core Sequence:NEY*FY*V
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: FGFR1 (flg), FGFR2 (bek, KGFR), FGFR3, and FGFR4. 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 FGFR1 can be phosphorylated: Tyr463, 583, 585, 653, 654, 730, and 766. Tyr653 and Tyr654 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).
1. Powers, C.J. et al. (2000) Endocr Relat Cancer 7, 165-97.
2. Reilly, J.F. et al. (2000) J Biol Chem 275, 7771-8.
3. Mohammadi, M. et al. (1996) Mol Cell Biol 16, 977-89.
4. Mohammadi, M. et al. (1991) Mol Cell Biol 11, 5068-78.
5. Larsson, H. et al. (1999) J Biol Chem 274, 25726-34.
Data Sheets & Documentation
For Research Use Only. Not For Use In Diagnostic Procedures. Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. DELFIA is a registered trademark of PerkinElmer, Inc.
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