Product Pathways - Tyrosine Kinase/ Adaptors

FGF Receptor 1 Kinase #7730

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 FGFR-1 kinase (Gly400-Arg820), 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 FGFR-1 (Gly400-Arg820) 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-FGFR-1 fusion protein was analyzed using SDS/PAGE followed by anti-FGFR-1 Western blot (A) or Silver stain (B).

Kinase Assay - Radiometric

Figure 2. FGFR-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, 5 µg/50 µl and 50 ng/50 µl Recombinant FGFR-1.

Kinase Assay - DELFIA

Figure 3. Dose dependence curve of FGFR-1 kinase activity: DELFIA® data generated using Phospho-Tyrosine mAb (P-Tyr-100) #9411 to detect phosphorylation of substrate peptide (#1315) by FGFR-1 kinase. In a 50 µl reaction, increasing amounts of FGFR-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-FGFR-1 fusion protein is 78 kDa. The purified kinase was quality controlled for purity using SDS-PAGE followed by Silver stain and Western blot [Fig.1]. FGFR-1 kinase activity was determined using a radiometric assay [Fig.2]. A kinase dose dependency assay was performed to measure FGFR-1 activity using HTScan™ FGF Receptor 1 Kinase Assay Kit #7420 [Fig.3].

Background

Fibroblast growth factors are a family of broad-spectrum growth factors influencing a plethora of cellular activities. The interaction of at least 23 ligands, 4 receptors and multiple coreceptors provides a dramatic complexity to a signaling system capable of effecting a multitude of responses (1,2). Basic fibroblast growth factor (bFGF or FGF2), initially identified as a mitogen with prominent angiogenic properties, is now recognized as a multifunctional growth factor (3). It is clear that bFGF produces its biological effects in target cells by signaling through cell-surface FGF receptors. bFGF binds to all four FGF receptors. Ligand binding induces receptor dimerization and autophosphorylation, allowing binding and activation of cytoplasmic downstream target proteins, including FRS-2, PLC and Crk (4,5). The FGF signaling pathway appears to play a significant role not only in normal cell growth regulation but also in tumor development and progression (6).Acidic FGF (aFGF or FGF1) is another extensively investigated protein of the FGF family. aFGF shares 55% DNA sequence homology with bFGF. These two growth factors are ubiquitously expressed and exhibit a wide spectrum of similiar biological activities with quantitative differences likely due to variation in receptor affinity or binding (7).

Application References

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Companion Products

7420 HTScan® FGF Receptor 1 Kinase Assay Kit
1315 PYK2 (Tyr402) 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.