Recombinant Mouse QSOX1/Quiescin Q6 Protein, CF Summary
with an N-terminal Met and 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 Tris and NaCl.|
|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 Buffer: 50 mM Sodium Phosphate, pH 7.5
- Recombinant Mouse QSOX1/Quiescin Q6 (rmQSOX1) (Catalog # 9257-QS)
- Coupling Enzyme: Horseradish Peroxidase (HRP), 250-330 U/mg (Sigma, Catalog # P8375), 250 units/mL stock in 0.1 M Sodium Phosphate, pH 8.0
- Substrate Component 1: Dithiothreitol (DTT) (Amresco, Catalog # 0281), 1 M stock in deionized water
- Substrate Component 2: Amplex® Ultra Red (AUR) (Invitrogen, Catalog # A36006), 10 mM stock in DMSO
- F15 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Dilute rmQSOX1 to 1 ng/μL in Assay Buffer.
- Prepare a Substrate Mixture containing 100 µM AUR, 2 units/mL HRP and 300 µM DTT in Assay Buffer. Make sure to add the DTT component right before reading the plate.
- Load 50 μL of 1 ng/μL rmQSOX1 into the plate, and start the reaction by adding 50 μL of Substrate Mixture. Include a Substrate Blank containing 50 μL of Assay Buffer and 50 μL of Substrate Mixture.
- Read at excitation and emission wavelengths of 544 nm and 590 nm in kinetic mode for 5 minutes. Note: A cutoff must be set at a wavelength of 570 nm.
- 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 a fluorescent standard prepared by incubating 50 µM AUR, 1 unit/mL HRP and 150 µM DTT and a curve of Hydrogen Peroxide (Sigma, Catalog # H1009) in Assay Buffer. Use this oxidized AUR curve to determine the conversion factor.Per Well:
- rmQSOX1: 0.05 μg
- DTT: 150 µM
- HRP: 1 unit/mL
- AUR: 50 µM
Background: QSOX1/Quiescin Q6
Sulfhydryl Oxidase-1 (QSOX1) is an enzyme that contains thioredoxin and sulfhydryl oxidase domains (1-3). It is synthesized with a C-terminal transmembrane segment, but soluble secreted forms can be generated by alternative splicing or proteolytic shedding within the Golgi (4). Within the region encompassing both enzymatic domains and the central region, mouse QSOX1 shares 79% and 92% aa sequence identity with human and rat QSOX1, respectively. It plays a role in nascent protein folding by mediating disulfide oxidation (4-6). This activity is required for Laminin incorporation into the extracellular matrix (7). QSOX1 is up-regulated in many cancers and supports tumor cell proliferation and invasion (1, 8).
- Lake, D.F. and D.O. Faigel (2014) Antioxid. Redox. Signal. 21:485.
- Kodali, V.K. and C. Thorpe (2010) Antioxid. Redox Signal. 13:1217.
- Matsuba, S. et al. (2002) J. Dermatol. Sci. 30:50.
- Rudolf, J. et al. (2013) Biochem. J. 454:181.
- Heckler, E.J. et al. (2008) Biochemistry 47:4955.
- Alon, A. et al. (2012) Nature 488:414.
- Ilani, T. et al. (2013) Science 341:74.
- Katchman, B.A. et al. (2011) Mol. Cancer Res. 9:1621.
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