详细说明
- Purity>90%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
- Endotoxin Level<1.0 EU per 1 μg of the protein by the LAL method.
- ActivityMeasured by its ability to phosphorylate 1-deoxy-1-morpholino-D-fructose. The specific activity is >30 pmol/min/μg, as measured under the described conditions. See Activity Assay Protocol on .
- SourceE. coli-derived Glu2-Lys309, with N-terminal Met and 6-His tag
- Accession #
- N-terminal Sequence
AnalysisMet - Predicted Molecular Mass36 kDa
- SDS-PAGE37 kDa, reducing conditions
9155-FU | | |
Formulation Supplied as a 0.2 μm filtered solution in Tris, NaCl, TCEP and Glycerol. | ||
Shipping The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below. | ||
Stability & Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
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- Universal Kinase Activity Kit (Catalog # )
- 10X Assay Buffer (supplied in kit): 250 mM HEPES, 1.5 M NaCl, 100 mM MgCl2, 100 mM CaCl2, pH 7.0
- Recombinant Human Fructosamine-3-kinase/FN3K (rhFN3K) (Catalog # 9155-FU)
Substrate: 1-Deoxy-1-morpholino-D-fructose (Sigma, Catalog # D6149), 40 mM stock in deionized water - 96-well Clear Plate (Catalog # )
- 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 in 1X Assay Buffer. The standard curve has a range of 0.078 to 5 nmol per well.
- Prepare a reaction mixture containing 0.4 mM ATP (supplied in kit) and 2 mM 1-Deoxy-1-morpholino-D-fructose in 1X Assay Buffer.
- Dilute rhFN3K to 33.3 µg/mL 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 1X Assay Buffer.
- Load 15 µL of 33.3 µg/mL rhFN3K into empty wells of the same plate as the curve. Include a control containing 15 µL of 1X Assay Buffer.
- Add 25 µL of reaction mixture to the wells, excluding the standard curve.
- Seal plate and incubate at 37 °C for 30 minutes.
- Dilute Coupling Phosphatase 4 (supplied in kit) to10 µg/mL in 1X Assay Buffer.
- Add 10 µL of 10 µg/mL Coupling Phosphatase 4 to wells containing enzyme and control, excluding the standard curve.
- Seal plate and incubate 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 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 incubation time of 35 minutes).
- rhFN3K: 0.5 µg
- Coupling Phosphatase 4: 0.1 µg
- ATP: 0.2 mM
- 1-Deoxy-1-morpholino-D-fructose: 1 mM
- References:
- Monnier, V.M. (1989) Prog. Clin. Biol. Res. 304:1.
- Roper, H, et al. (1994). Carbohydr. Res. 262:257.
- Carson, J.F. et al. (1955). J. Am. Chem. So.c 77:1881.
- Reddy, S, et al. (1995) Biochemistry 34:10872.
- Bucala, R. and Cerami, A. (1992) Adv. Pharmacol. 23:1.
- Lyons, T.J. and Jenkins, A.J: (1997). Diabetes Rev. 5:365.
- Furusyo, N and Hayashi, J. (2013) Biochim. Biophys. Acta. 1830:5509.
- Lal, S, et al.(1993) J. Biol. Chem. 268:7763.
- Szwergold, B.S. et al. (2001) DIABETES 50:2139.
- Delpierre, G. et al. (2002) Biochem. J. 365: 801.
- Caruso, M.G. et al (2007) Oncology 73(1-2):72.
- Delplanque, J. et al. (2004) J. Biol.Chem. 279:46606.
- Petersen A, et al. (1992) Biochem J 284:363.
- Wu, Z.L. et al. (2011) PloS ONE 6(8):e23172.
- Entrez Gene IDs:64122 (Human); 63828 (Mouse)
- Alternate Names:EC 2.7.1.-; FN3K; fructosamine 3 kinase; Fructosamine-3-kinase