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.
Activity
Measured 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 .
Source
E. coli-derived Cys2-Gln382, with an N-terminal Met and 6-His tag
Accession #
N-terminal Sequence
AnalysisMet
Predicted Molecular Mass
44 kDa
SDS-PAGE
40 kDa, reducing conditions
5080-NM |
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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.
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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
