Recombinant Human FGFR1 Kinase Domain Protein, CF Summary
Product Specifications
Met399-Arg822 with an N-terminal Met and 6-His tag
Analysis
Product Datasheets
Carrier Free
CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.
In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.
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.
|
Assay Procedure
- 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
- Prepare 1X Assay Buffer by diluting 10X stock 10 fold with deionized water.
- 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.
- 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.
- Dilute ATP (supplied in kit) to 0.4 mM in 1X Assay Buffer.
- Dilute rhFGF R1 to 66.67 ng/µL in 1X Assay Buffer.
- Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.
- 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.
- Add 25 µL of 0.4 mM ATP to all wells, excluding the standard curve.
- Seal plate and incubate at 37 °C for 1 hour.
- Dilute Coupling Phosphatase 4 to 10 ng/µL in Assay Buffer.
- Add 10 µL of 10 ng/µL Coupling Phosphatase 4 to all wells, excluding the standard curve.
- Seal and incubate plate at room temperature for 5 minutes.
- Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.
- Add 100 µL of deionized water to all wells. Mix briefly.
- Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate sealed plate for 20 minutes at room temperature.
- Read plate at 620 nm (absorbance) in endpoint mode.
- 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
- rhFGF R1: 1.0 µg
- Coupling Phosphatase 4: 0.1 µg
- ATP: 0.2 mM
Reconstitution Calculator
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).
- Johnson, D.E. et al. (1990) Mol. Cell. Biol. 10:4728.
- Turner, N and Grose, R. (2010) Nat. Rev. Cancer 10:116.
- Pantoliano, M.W. et al (1994) Biochemistry 33:10229.
- Ornitz, D.M. et al. (996) J. Biol. Chem. 271:15292.
- Eswarakumar, V.P. et al. (2005) Cytokine Growth Factor Rev. 16:139.
- Peters, K.G. et al. (1992) Nature 358:678.
- Mohammadi, M. et al. (1996) Mol. Cell. Biol. 16:977.
- Mohammadi, M et al (1997) Science 276:955.
- Wu, Z.L. (2011) PloS ONE 6:e23172.
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