Recombinant Influenza A Virus H3N2 Neuraminidase Protein, CF
Recombinant Influenza A Virus H3N2 Neuraminidase Protein, CF Summary
Product Specifications
Ala46-Ile469, vasodilator-stimulated phosphoprotein tetramerization domain and a C-terminal 6-His tag
Analysis
Product Datasheets
6875-NM
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.
6875-NM
| Formulation | Supplied as a 0.2 μm filtered solution in Tris, NaCl, CaCl2, DTT and Glycerol. |
| Shipping | The product is shipped with dry ice or equivalent. 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: 25 mM MES, 500 mM NaCl, 5 mM CaCl2, pH 6.5
- Recombinant Influenza A Virus H3N2 Neuraminidase (rvH3N2) (Catalog # 6875-NM)
- Substrate: 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 (SpectraMax Gemini EM by Molecular Devices) or equivalent
- Dilute rvH3N2 Neuraminidase to 0.2 ng/μL in Assay Buffer.
- Dilute Substrate to 400 µM in Assay Buffer.
- Load 50 µL of the 0.2 ng/μL rvH3N2 Neuraminidase into a black well plate and start the reaction by adding 50 µL of 400 µM Substrate. Include a Substrate Blank containing Assay Buffer in place of rvH3N2 Neuraminidase.
- 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 (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).
- rvH3N2 Neuraminidase: 0.010 µg
- Substrate: 200 µM
Background: Viral Neuraminidase
Neuraminidase (NA) and hemagglutinin (HA) are the two predominant membrane glycoproteins found on the surface of an influenza virus particle. They are essential for the infectious cycle of the virus. HA recognizes and binds to the sialic acid on the host cell membrane to initiate a viral infection. NA cleaves the sialic acid at the end of the cycle, allowing the progeny virus to leave the host thus initiating the next round of infection (1). In the early stage of an infection, NA may also assist in viral penetration of the mucus layer in the airway of a host. Nine subtypes of NA (N1 to N9) have been identified, all of which are believed to be tetrameric and share a basic structure consisting of a globular head, a thin stalk region, and a small hydrophobic region that anchors the protein in the virus membrane (2). Glycosylation is also found to be important for the stability and activity of these enzymes (3). Due to their critical role in the infectious cycle of a virus, influenza viral neuraminidases are frequently used as targets for drug design. Both the anti-influenza drugs Tamiflu® and Relenza® are neuraminidase inhibitors. According to a recent structure determination (4), neuraminidases from influenza type A viruses form two genetically distinct groups, with the N1 and N2 neuraminidases representing each of the two groups. This recombinant protein is based on the sequences of the virus isolated from 1968 Hong Kong flu pandemic (5). An artificial tetramerization domain from the vasodilator-stimulated phosphoprotein (6) was inserted at the N-terminus to assist in oligomerization of the recombinant protein.
- Palese, P. & Compans, R. W. (1976) J. Gen. Virol. 33:159.
- Colman, P. M. et al. (1983) Nature 303:41.
- Wu, Z.L. et al. (2009) Biochem. Biophys. Res. Commun. 379:749.
- Russell, R.J. et al. (2006) Nature 443:45.
- Shortridge, K.F. et al. (1977) Science 196:1454.
- Kuhnel, K. et al. (2004) Proc. Natl. Acad. Sci. U. S. A. 101:17027.
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