Recombinant Human NQO-1 Protein, CF
Recombinant Human NQO-1 Protein, CF Summary
Met1-Lys274, with a C-terminal 6-His tag
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.
|Formulation||Supplied as a 0.2 μm filtered solution in NaH2PO4 and NaCl.|
|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 Buffer: 50 mM HEPES, 0.2 M NaCl, 5 µM FAD, 0.05% Tween® 20, pH 7.5
- Recombinant Human NQO-1 (rhNQO-1) (Catalog # 7567-DH)
- beta -Nicotinamide Adenine Dinucleotide, reduced (NADH) (Sigma, Catalog # N8129), 20 mM stock in 0.1 M Sodium Borate, pH 9.0
- Resazurin (Catalog # AR002) (MW = 251.17 Da)
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Dilute rhNQO-1 to 0.0075 µg/mL in Assay Buffer.
- Dilute NADH to 800 µM in Assay Buffer.
- Dilute Resazurin to 40 µM in Assay Buffer.
- Combine equivalent volumes of diluted NADH and Resazurin to make the Substrate Mixture. Mix well and use immediately.
- Load 50 µL of 0.0075 µg/mL rhNQO-1 into a plate, and start the reaction by adding 50 µL of the Substrate Mixture. For a Substrate Blank, load 50 µL of Assay Buffer and 50 µL of Substrate Mixture.
- Read at excitation and emission wavelengths of 540 nm and 585 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 Resorufin (Sigma, Catalog # R3257).
- rhNQO-1: 0.375 ng
- NADH: 200 µM
- Resazurin: 10 µM
NAD(P)H:quinone acceptor oxidoreductase 1 (NQO1), also known as DT-diaphorase, is a widely-distributed FAD-dependent flavoprotein that promotes 2-electron reductions of quinones, quinoneimines, nitroaromatics, and azo dyes (1, 2). As a result it prevents the one electron reduction of quinones that results in the production of radical species. NQO1 is a highly-inducible enzyme that is regulated by the Keap1/Nrf2/ARE pathway (2, 3). The increase and decrease of NQO1 levels are associated with decreased and increased susceptibilities to oxidative stress, respectively. Thus, NQO1 is a marker cytoprotective enzyme in oxidative stress. Independently of its catalytic function, NQO1 plays a role in regulating the proteosomal degradation of p53, p73a, and p33 (2). NQO1 physically interacts with p53 and p73 in an NADH-dependent manner and protects them from 20S proteasomal degradation in a ubiquitin independent pathway (4).
- Gong, X. et al. (2008) Vitamin Horm. 78:85.
- Dinkova-Kostova, A. T., and Talalay, P. (2010) Arch. Biochem. Biophys. 501:116.
- Hayes, J. D. and McMahon, M. (2009) Trends Biochem. Sci. 34:176.
- Asher, G. et al. (2005) Genes Dev. 19:316.
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