Recombinant Human Spinesin Protein, CF Summary
Tyr71-Leu457 (Phe369Leu), with an N-terminal 9-His tag
Accession # NP_110397
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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 MES 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.
- Activation Buffer: 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
- Assay Buffer: 50 mM Tris, pH 8.0
- Recombinant Human Spinesin (rhSpinesin) (Catalog # 2495-SE)
- Bacterial Thermolysin (Thermolysin) (Catalog # 3097-ZN)
- 1,10 Phenanthroline (Sigma, Catalog # 320056), 0.6 M stock in DMSO
- Substrate: BOC-Gln-Ala-Arg-AMC (Catalog # ES014), 10 mM stock in DMSO
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Dilute rhSpinesin to 200 µg/mL in Activation Buffer.
- Dilute Thermolysin to 2 µg/mL in Activation Buffer.
- Combine equal amounts of 200 µg/mL rhSpinesin and 2 µg/mL Thermolysin.
- Incubate the reactions at 37 °C for 1 hour.
- Stop the Thermolysin activity by adding 1,10 Phenanthroline to a final concentration of 10 mM.
- Dilute activated rhSpinesin to 2 ng/µL in Assay Buffer.
- Dilute Substrate to 200 µM in Assay Buffer.
- Load 50 µL of 2 ng/µL rhSpinesin into a plate, and start the reaction by adding 50 µL of 200 µM Substrate.
- Include a Substrate Blank containing 50 µL of Assay Buffer and 50 µL of Substrate.
- Read at excitation and emission wavelengths of 380 nm and 460 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 7-Amino, 4-Methyl Coumarin (Sigma, Catalog # A-9891).
- rhSpinesin: 0.100 µg
- Thermolysin: 0.001 µg
- Substrate: 100 µM
Spinesin, encoded by the TMPRSS5 gene, is a member of type II transmembrane serine proteases (TTSPs) (1). Human Spinesin contains the following structural domains: a short N-terminal cytoplasmic tail (amino acid residues 1-49), a transmembrane domain (residues 50-70), a stem region and a serine protease domain (residues 71-457) (2). The domain structure of Spinesin is common to other TTSPs, many of which have additional domains. The stem region of Spinesin contains a scavenger receptor-like domain. The ectodomain of human Spinesin (residues 71-457) was expressed and purified as a single chain pro-enzyme. The deduced amino acid sequence contains a Leu instead of a Phe residue at position 369; the former is identical to the mouse protein (3, 4). The pro-enzyme can be activated and the resulting enzyme activity can be measured as described in the Activity Assay Protocol.
- Hooper, J.D. et al. (2001) J. Biol. Chem. 276:857.
- Yamaguchi, Y. et al. (2002) J. Biol. Chem. 277:6806.
- Carninci, P. et al. (2000) Genome Res. 10:1617.
- Shibata, K. et al. (2000) Genome Res. 10:1757.
Citation for Recombinant Human Spinesin 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.
1 Citation: Showing 1 - 1
Prostasin is required for matriptase activation in intestinal epithelial cells to regulate closure of the paracellular pathway.
Authors: Buzza M, Martin E, Driesbaugh K, Desilets A, Leduc R, Antalis T
J Biol Chem, 2013;288(15):10328-37.
Sample Types: Whole Cells
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