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Recombinant Influenza A Virus H1N1 Neuraminidase Protein, CF

Catalog #: 4858-NM
2 Citations Datasheet
Catalog # Availability Size / Price Qty
4858-NM-005
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Recombinant Influenza A Virus H1N1 Neuraminidase Protein, CF Summary

Product Specifications

Purity
>80%, 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 >2,500 pmol/min/µg, as measured under the described conditions.
Source
Spodoptera frugiperda, Sf 21 (baculovirus)-derived influenza a virus h1n1 Viral Neuraminidase protein
Ser37-Lys469, with an N-terminal 6-His tag
Accession #
AAF77036
N-terminal Sequence
Analysis
His
Predicted Molecular Mass
48 kDa
SDS-PAGE
57 kDa, reducing conditions

Product Datasheets

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4858-NM

Product Datasheet

Carrier Free

What does CF mean?

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.

What formulation is right for me?

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.

4858-NM

Formulation Supplied as a 0.2 μm filtered solution in Tris, NaCl and Glycerol.
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.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.

Assay Procedure

Materials
  • Assay Buffer: 50 mM Tris, 5 mM CaCl2, 200 mM NaCl, pH 7.5
  • Recombinant Influenza A Virus H1N1 Neuraminidase (rH1N1 Neuraminidase) (Catalog # 4858-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
  1. Dilute rH1N1 Neuraminidase to 0.05 ng/µL in Assay Buffer.
  2. Dilute Substrate to 400 µM in Assay Buffer.
  3. Load 50 µL of 0.05 ng/µL rH1N1 Neuraminidase into the plate, and start the reaction by adding 50 µL of 400 µM Substrate. As a Substrate Blank load 50 µL of Assay Buffer and 50 µL of Substrate.
  4. Read at excitation and emission wavelengths of 365 nm and 445 nm (top read), respectively in kinetic mode for 5 minutes.
  5. Calculate specific activity:

     Specific Activity (pmol/min/µg) =

 Adjusted Vmax* (RFU/min) x Conversion Factor** (pmol/RFU)
amount of enzyme (µg)

     *Adjusted for Substrate Blank
     **Derived using calibration standard 4-Methylumbelliferone (Sigma, Catalog # M1381).

Per Well:
  • rH1N1 Neuraminidase: 0.0025 µg
  • Substrate: 200 µM

Background: Viral Neuraminidase

Neuraminidase (NA) and hemagglutinin (HA) are major membrane glycoproteins found on the surface of influenza virus. HA is a lectin that binds sialic acid on host cell membrane. NA is a sialic acid hydrolase that specifically clips off terminally located sialic acid on host cell surface. The two proteins are essential for the infectious cycle of the influenza virus. During initial infection, an influenza virus will hold onto an epithelial cell through HA-sialic acid interaction. At the end of an infectious cycle, the NA will cleave the sialic acid on the host cell membrane, releasing the formed viral particle from the HA-sialic acid bondage (1). The neuraminidase activity is also thought to help the virus penetrate mucus. Nine subtypes of NA have been identified, all of which are tetrameric and share a common structure consisting of a globular head, a thin stalk region, and a small hydrophobic region that anchors the protein in the virus membrane (2). The purified rvH1N1NA consists of amino acid residues 37 to 469 as deduced from the 1918 Spanish flu virus NA (A/Bervig_Mission/1/18) (3). It has a distinct N-glycan profile and is resistant to trypsin digestion (4).

References
  1. Palese, P. & Compans, R. W. (1976) J. Gen. Virol. 33:159.
  2. Colman, P. M. et al. (1983) Nature 303:41.
  3. Reid, A. (2000) Proc. Natl. Acad. Sci. USA 97:6785.
  4. Wu, Z.L. et al. (2009) Biochem. Biophys. Res. Comm. 379:749.
Alternate Names
NANH; Sialidase; Viral Neuraminidase

Citations for Recombinant Influenza A Virus H1N1 Neuraminidase Protein, CF

R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.

2 Citations: Showing 1 - 2
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  1. Enzymatic synthesis and properties of glycoconjugates with legionaminic acid as a replacement for neuraminic acid.
    Authors: Watson DC, Leclerc S, Wakarchuk WW, Young NM
    Glycobiology, 2010-10-25;21(1):99-108.
    Applications: Enzyme Assay
  2. Active 1918 pandemic flu viral neuraminidase has distinct N-glycan profile and is resistant to trypsin digestion.
    Authors: Wu ZL, Ethen C, Hickey GE, Jiang W
    Biochem. Biophys. Res. Commun., 2009-01-06;379(3):749-53.
    Species: Virus
    Sample Types: Recombinant Protein
    Applications: Enzyme Assay

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