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 >80,000 pmol/min/µg, as measured under the described conditions. See Activity Assay Protocol on .
- SourceE. coli-derived Cys2-Gln382, with an N-terminal Met and 6-His tag
- Accession #
- N-terminal Sequence
AnalysisMet - Predicted Molecular Mass44 kDa
- SDS-PAGE40 kDa, reducing conditions
| 5080-NM | | |
| Formulation Supplied as a 0.2 μm filtered solution in Tris, NaCl and EDTA. | ||
| 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 MES, 100 mM NaCl, 0.05% (w/v) Brij-35, pH 6.5
- Recombinant C. perfringens Neuraminidase (rCpNA) (Catalog # 5080-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 rCpNA to 0.04 ng/µL in Assay Buffer.
- Dilute Substrate to 400 µM in Assay Buffer.
- Load 50 µL of 0.04 ng/µL rCpNA into a plate, and start the reaction by adding 50 µL of 400 µM Substrate.
- 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).
Per Well:- rCpNA: 0.002 µg
- Substrate: 200 µM
Background: Bacterial Neuraminidase
Neuraminidase and Sialidase are the common names for N‑acetyl‑neuraminyl hydrolase (1-3). The enzyme catalyzes the hydrolysis of alpha 2-3 and alpha 2-6 linked N‑acetyl‑neuraminic acid residues from glycoproteins and glycolipids. It has little activity against the alpha 2-8 linked N‑acetyl‑neuraminic acid residues.
- References:
- Christensen, S. et al. (2005) Biotechnol. Appl. Biochem. 41:225.
- Roggentin, P. et al. (1988) FEBS Lett. 238:31.
- Corfield, A. et al. (1983) Biochim. Biophys. Acta 744:121.
- Alternate Names:Bacterial Neuraminidase
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