Recombinant Mouse Spinesin Protein, CF Summary
Product Specifications
Tyr61-Arg445, with an N-terminal 7-His tag
Accession # NP_109634
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.
1928-SE
| 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.
|
Assay Procedure
- Activation Buffer: 50 mM Tris, 10 mM CaCl2, pH 7.5
- Assay Buffer: 100 mM Tris, pH 8.5
- Recombinant Mouse Spinesin (rmSpinesin) (Catalog # 1928-SE)
- Trypsin (Sigma, Catalog # T-1426)
- Recombinant Human Serpin A1/ alpha 1‑Antitrypsin (rhSerpin A1) (Catalog # 1268-PI)
- Fluorogenic Peptide Substrate: BOC-Gln-Ala-Arg-AMC (Catalog # ES014)
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Dilute rmSpinesin to 200 µg/mL in Activation Buffer.
- Dilute Trypsin to 0.4 µg/mL in Activation Buffer.
- Combine equal volumes of 200 µg/mL rmSpinesin and 0.4 µg/mL Trypsin. Include a Trypsin control containing Activation Buffer in place of rmSpinesin.
- Incubate at room temperature for 1 hour.
- Dilute rhSerpin A1 to 36 µg/mL in Assay Buffer.
- Dilute the reaction mixtures in half by adding rhSerpin A1 for final concentrations of 18 µg/mL rhSerpin A1, 50 µg/mL rmSpinesin, and 0.1 µg/mL Trypsin. Addition of rhSerpin A1 will stop the activity of Trypsin. Control from step 3 will verify that rhSerpin A1 is working properly.
- Dilute rmSpinesin to 2 ng/µL in Assay Buffer. Dilute Trypsin control equally.
- Dilute Substrate to 200 µM in Assay Buffer.
- Load 50 µL of the diluted samples into a black well plate, and start the reaction by adding 50 µL of 200 µM Substrate. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of 200 µM Substrate.
- Read at excitation and emission wavelengths of 380 nm and 460 nm, 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 (AMC) (Sigma, Catalog # A-9891).
- rmSpinesin: 0.1 µg
- Substrate: 100 µM
Reconstitution Calculator
Background: Spinesin
Spinesin, encoded by the TMPRSS5 gene, is a new member of type II transmembrane serine proteases (TTSPs) (1). Mouse Spinesin contains the following structural domains: a short N-terminal cytoplasmic tail, a transmembrane domain, a stem region and a serine protease domain (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. There could be 4 types of transcripts due to alternative splicing (3). Type 4 predicts 10 extra amino acids at the N-terminus as compared to type 3. The ectodomain corresponding to type 3 (residues 61‑445) or type 4 (residues 71‑455) was expressed and purified as a single chain pro-enzyme. By SDS‑PAGE, the pro‑enzyme migrates as multiple forms, possibly due to differential glycosylation. The pro‑enzyme can be activated by trypsin treatment. The resulting enzyme is active and its activity is measured as described above. The activated enzyme is a disulfide bond‑linked dimer.
- Shibata, K. et al. (2000) Genome Res. 10:1757.
- Yamaguchi, Y. et al. (2002) J. Biol. Chem. 277:6806.
- Watanable, Y. et al. (2004) Biochem. Biophys. Res. Commun. 324:333.
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