详细说明
- Purity>95%, by SDS-PAGE under reducing conditions and visualized by silver stain
- Endotoxin Level<1.0 EU per 1 μg of the protein by the LAL method.
- ActivityMeasured by its ability to cleave a fluorogenic substrate, 2’-(4-Methylumbelliferyl)-alpha -D-N-acetylneuraminic acid. The specific activity is >25,000 pmol/min/µg, as measured under the described conditions. See Activity Assay Protocol on .
- SourceE. coli-derived Pro42-Gly403, with an N-terminal Met and 6-His tag Accession # BAA00852
- Accession #
- N-terminal Sequence
AnalysisMet - Predicted Molecular Mass40 kDa
- SDS-PAGE42 kDa, reducing conditions
| 5084-NM | | |
| Formulation Supplied as a 0.2 μm filtered solution in Tris and NaCl. | ||
| 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
Materials
- Assay Buffer: 50 mM Sodium Acetate, 150 mM NaCl, pH 4.5
- Recombinant M. viridifaciens Neuraminidase (rMvNA) (Catalog # 5084-NM)
- Substrate: 2’-(4-Methylumbelliferyl)-alpha -D-N-acetylneuraminic acid (Sigma, Catalog # M8639), 10 mM stock in DMSO
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Dilute rMvNA to 0.2 ng/µL in Assay Buffer.
- Dilute Substrate to 400 µM with Assay Buffer.
- Load into plate 50 µL of 0.2 ng/µL rMvNA and start the reaction by adding 50 µL of 400 µM Substrate. Include a Substrate Blank containing 50 µL of Substrate and 50 µL of Assay Buffer.
- Read at excitation and emission wavelengths of 365 nm and 445 nm (top read), respectively, in kinetic mode for 5 minutes.
- Calculate specific activity:
| Specific Activity (pmoles/min/µg) = | Adjusted Vmax* (RFU/min) x Conversion Factor** (pmole/RFU) |
| amount of enzyme (µg) |
*Adjusted for Substrate Blank
**Derived using calibration standard 4-Methylumbelliferone (Sigma, Catalog # M1381).
- rMvNA: 0.010 µg
- Substrate: 200 µM
Background: Bacterial Neuraminidase
Neuraminidase is the common name for N-acetyl-neuraminyl hydrolase (Sialidase). Micromonospora viridifaciens Neuraminidase catalyzes the hydrolysis of alpha 2-3 and alpha 2-6 and alpha 2-8 linked N-acetyl-neuraminic acid residues from glycoproteins and glycolipids (1). The architecture of the full-length protein includes a canonical neuraminidase enzymatic domain, a linker domain and a C-terminal galactose binding domain (2). The full-length protein contains 647 amino acids and has a molecular weight of 68 kDa. It is easily degraded to 52 kDa and 41 kDa fragments (3). The expressed enzyme only contains the enzymatic domain.
- References:
- Aisaka, K. & Uwajima, T. (1987) FEBS Microbiol. Lett. 44:289.
- Gaskell, A. et al. (1995) Structure 3:1197.
- Sakurada, K. et al. (1992) J. Bacteriol. 174:6896.
- Alternate Names:Bacterial Neuraminidase
400-6226-992