Recombinant Human QSOX1/Quiescin Q6 Protein, CF Summary
Product Specifications
Ser33-Ala546, with an N-terminal Met and 6-His tag
Analysis
Product Datasheets
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.
9209-QS
| 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 Procedure
- Assay Buffer: 50 mM Sodium Phosphate, pH 7.5
- Recombinant Human QSOX1/Quiescin Q6 (rhQSOX1) (Catalog # 9209-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 rhQSOX1 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 DTT to the Substrate Mixture right before loading the plate.
- Load 50 μL of 1 ng/μL rhQSOX1 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.
- rhQSOX1: 0.05 μg
- DTT: 150 µM
- HRP: 1 unit/mL
- AUR: 50 µM
Reconstitution Calculator
Background: QSOX1/Quiescin Q6
Sulfhydryl Oxidase-1 (QSOX1) is an approximately 80 kDa 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, human QSOX1 shares 79% aa sequence identity with mouse and rat QSOX1. It plays a role 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.
- Coppock, D.L. et al. (1998) Genomics 54:460.
- 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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