Recombinant Human FGFR1 Kinase Domain Protein, CF

R&D Systems | Catalog # 8657-FR

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Key Product Details

  • R&D Systems Sf 21 (baculovirus)-derived Recombinant Human FGFR1 Kinase Domain Protein (8657-FR)
  • Quality control testing to verify active proteins with lot specific assays by in-house scientists
  • All R&D Systems proteins are covered with a 100% guarantee

Source

Sf 21 (baculovirus)

Accession Number

P11362

Applications

Enzyme Activity
View all FGFR1 Proteins and Enzymes »

Product Specifications

Source

Spodoptera frugiperda, Sf 21 (baculovirus)-derived human FGF R1 protein
Met399-Arg822 with an N-terminal Met and 6-His tag

Purity

>90%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane.

Endotoxin Level

<1.0 EU per 1 μg of the protein by the LAL method.

N-terminal Sequence Analysis

No results obtained: Met predicted. Protein identity confirmed by MS analysis of tryptic fragments.

Predicted Molecular Mass

49 kDa

SDS-PAGE

45-58 kDa, reducing conditions

Activity

Measured by its ability to hydrolyze the 5’-phosphate groups from the substrate adenosine-5’-triphosphate (ATP). The orthophosphate product is measured by a Malachite Green Phosphate Detection Kit (Catalog # DY996).
The specific activity is >12 pmol/min/μg, as measured under the described conditions.

Formulation, Preparation, and Storage

8657-FR
Formulation Supplied as a 0.2 μm filtered solution in Tris, NaCl and TCEP.
Shipping The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.
Stability & Storage Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.

Background: FGFR1

Fibroblast growth factor receptors (FGF Rs) are single-pass transmembrane proteins with extracellular fibroblast growth factor (FGF) binding domains and cytoplasmic tyrosine-protein kinase domains (1). FGF Rs play essential roles in the regulation of embryonic development, cell proliferation, differentiation and migration (2). Upon FGF and heparan sulfate binding, the FGF Rs form homodimers and the kinase domains are activated (3, 4). The activated kinases then phosphorylate downstream effector molecules such as PLC-g1, FRS2, Gab1, and SHB, leading to the activation of several signaling cascades (5). For example, activation of PLC-g1 leads to the production of the cellular signaling molecules diacylglycerol and inositol 1,4,5-trisphosphate (6). Phosphorylation of FRS2 triggers recruitment of GRB2, Gab1, PI 3-Kinase p85, and SOS1, activation of Ras, ERK2, and ERK1, and activation of the MAP kinase and AKT1 signaling pathways (7). In this construct, the kinase domain of human FGF R1 is expressed as a constitutively active enzyme (8) and the activity was assayed using a phosphatase-coupled method (9).

References

  1. Johnson, D.E. et al. (1990) Mol. Cell. Biol. 10:4728.
  2. Turner, N and Grose, R. (2010) Nat. Rev. Cancer 10:116.
  3. Pantoliano, M.W. et al (1994) Biochemistry 33:10229.
  4. Ornitz, D.M. et al. (996) J. Biol. Chem. 271:15292.
  5. Eswarakumar, V.P. et al. (2005) Cytokine Growth Factor Rev. 16:139.
  6. Peters, K.G. et al. (1992) Nature 358:678.
  7. Mohammadi, M. et al. (1996) Mol. Cell. Biol. 16:977.
  8. Mohammadi, M et al (1997) Science 276:955.
  9. Wu, Z.L. (2011) PloS ONE 6:e23172.

Long Name

Fibroblast Growth Factor Receptor 1

Alternate Names

CD331, FGF R1, Flt-2

Entrez Gene IDs

2260 (Human); 14182 (Mouse)

Gene Symbol

FGFR1

UniProt

P11362

Additional FGFR1 Products

Product Documents for Recombinant Human FGFR1 Kinase Domain Protein, CF

Certificate of Analysis

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Product Specific Notices for Recombinant Human FGFR1 Kinase Domain Protein, CF

For research use only

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Protocols

View specific protocols for Recombinant Human FGFR1 Kinase Domain Protein, CF (8657-FR):

Materials
  • Universal Kinase Activity Kit (Catalog # EA004)
  • 10X Assay Buffer (supplied in kit): 250 mM HEPES, 1.5 M NaCl, 100 mM MgCl2, 100 mM CaCl2, pH 7.0
  • Recombinant human FGF R1 (rhFGF R1) (Catalog # 8657-FR)
  • ATP (supplied in kit): 10 mM
  • 96-well Clear Plate (Catalog # DY990)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Prepare 1X Assay Buffer by diluting 10X stock 10 fold with deionized water.
  2. Dilute 1 mM Phosphate Standard provided by the Universal Kinase Activity Kit by adding 40 µL of the 1 mM Phosphate Standard to 360 µL of 1X Assay Buffer for a 100 µM stock. This is the first point of the standard curve.
  3. Complete the standard curve by performing six one-half serial dilutions of the 100 µM Phosphate stock using 1X Assay Buffer. The standard curve has a range of 0.078 to 5 nmol per well.
  4. Dilute ATP (supplied in kit) to 0.4 mM in 1X Assay Buffer.
  5. Dilute rhFGF R1 to 66.67 ng/µL in 1X Assay Buffer.
  6. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.
  7. Load 15 µL of 66.67 ng/µL rhFGF R1 into empty wells of the same plate as the curve. Include a Control containing 15 μL of 1X Assay Buffer.
  8. Add 25 µL of 0.4 mM ATP to all wells, excluding the standard curve. 
  9. Seal plate and incubate at 37 °C for 1 hour.
  10. Dilute Coupling Phosphatase 4 to 10 ng/µL in Assay Buffer.
  11. Add 10 µL of 10 ng/µL Coupling Phosphatase 4 to all wells, excluding the standard curve.
  12. Seal and incubate plate at room temperature for 5 minutes.
  13. Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.
  14. Add 100 µL of deionized water to all wells. Mix briefly.
  15. Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate sealed plate for 20 minutes at room temperature.
  16. Read plate at 620 nm (absorbance) in endpoint mode.
  17. Calculate specific activity:

     Specific Activity (pmol/min/µg) =

 Phosphate released* (nmol) x (1000 pmol/nmol)
Incubation time (min) x amount of enzyme (µg)

     *Derived from the phosphate standard curve using linear or 4-parameter fitting and adjusted for Control
     **Decoupled reaction (use 65 minutes for incubation time); no coupling rate constant

Per Reaction:

  • rhFGF R1:  1.0 µg
  • Coupling Phosphatase 4:  0.1 µg
  • ATP:  0.2 mM

FAQs

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