Recombinant Human N-Acetylmannosamine Kinase/GNE Protein, CF
Recombinant Human N-Acetylmannosamine Kinase/GNE Protein, CF Summary
Product Specifications
Thr406-Arg720, 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.
8268-GK
| Formulation | Supplied as a 0.2 μm filtered solution in Tris, NaCl and DTT. |
| 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
- Assay Buffer (10X): 250 mM HEPES, 1500 mM NaCl, 100 mM MgCl2, 100 mM CaCl2 pH 7.0 (supplied in kit)
- Recombinant Human N‑Acetylmannosamine Kinase/GNE (rhGNE) (Catalog # 8268-GK)
- N-Acetyl-D-mannosamine (Sigma, Catalog # A8176), 100 mM stock in deionized water
- Universal Kinase Activity Kit (Catalog # EA004)
- 96-well Clear Plate (Catalog # DY990)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Prepare 1X Assay Buffer by diluting 10X Assay Buffer using 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.
- Prepare standard curve by performing seven one-half serial dilutions of the 100 µM Phosphate stock in 1X Assay Buffer. The standard curve has a range of 0.039 to 2.5 nmol per well.
- Load 50 µL of each dilution of the standard curve into a plate in triplicate. Include a curve blank containing 50 μL of 1X Assay Buffer.
- Prepare Substrate Mixture composed of 0.4 mM ATP and 2 mM N-Acetyl-D-mannosamine in 1X Assay Buffer.
- Dilute rhGNE to 66.67 μg/mL in 1X Assay Buffer.
- Load 15 µL of the 66.67 μg/mL rhGNE into the plate in triplicate. Include a control containing 15 µL of 1X Assay Buffer.
- Dilute Coupling Phosphatase 4 (supplied in kit) to 10 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.
- Add 25 µL of Substrate Mixture to the wells, excluding the standard curve.
- Incubate sealed plate at room temperature for 10 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) x coupling rate** |
*Derived from the phosphate standard curve using linear fitting and adjusted for Control
**The coupling rate is 0.475 under these conditions.
- rhGNE: 1 µg
- Coupling Phosphatase 4: 0.1 µg
- ATP: 0.2 mM
- N-Acetyl-D-mannosamine: 1 mM
Reconstitution Calculator
Background: N-Acetylmannosamine Kinase/GNE
Sialic acid modification of cell surface molecules is crucial for many biologic processes, such as cell adhesion (1), formation or masking of recognition determinants (2), and stabilization of glycoprotein structure of glycoproteins (3). Furthermore, sialic acids are over-expressed in many tumor cells (4) and are associated with a higher metastatic potential (5). The rate limiting enzyme of sialic acid biosynthesis is a bifunctional enzyme, UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (GNE), that converts UDP-GlcNAc to ManNAc and subsequently phosphorylates the ManNAc to ManNAc 6-phosphate (6). The activity of GNE is regulated through feedback inhibition by the downstream product, CMP-sialic acid (7). In humans, GNE mutation causes two genetic diseases, sialuria (8) and hereditary inclusion body myopathy (HIBM). In sialuria, a mutation of either Arg-263 or Arg-266 in the epimerase domain impairs the feedback inhibition of GNE (9). HIBM is an autosomal recessive neuromuscular disorder caused by more than 60 different point mutations (10).The recombinant human N-Acetylmannosamine Kinase only contains the kinase domain (11). The kinase activity was measured using a phosphatase-coupled method (12).
- Varki, A. (2007) Nature 446:1023.
- Karlsson, K.-A. (1995) Curr. Opin. Struct. Biol. 5:622.
- Rens-Domiano, S. and Reisine, T. (1991) J. Biol. Chem. 266:20094.
- Roth, J. et al. (1988) Proc. Natl. Acad. Sci. U. S. A. 85:2999.
- Bresalier, R. S. et al. (1990) Cancer Res. 50:1299.
- Stasche, R. et al. (1997) J. Biol. Chem. 273: 24319.
- Kornfeld, S. et al. (1964) Proc. Natl.Acad. Sci. U.S.A. 52:371.
- Weiss, P. et al. (1989) J. Biol. Chem. 264:17635.
- Eisenberg, I. et al. (2001) Nat. Genet. 29:83.
- Huizing, M. and Krasnewich, D. M. (2009) Biochim. Biophys. Acta 1792:881.
- Martinez, J. et al. (2012) J. Biol. Chem. 287:13656.
- Wu, Z. (2011) PLoS ONE 6:e23172.
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