Recombinant Mouse Furin Protein, CF Summary
Product Specifications
Gln25-Glu714, with a C-terminal 10‑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.
6450-SE
| Formulation | Supplied as a 0.2 μm filtered solution in Tris, CaCl2, NaCl, Brij-35 and Glycerol. |
| 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: 25 mM Tris, 1 mM CaCl2, 0.5% (w/v) Brij-35, pH 9.0
- Recombinant Mouse Furin (rmFurin) (Catalog # 6450-SE)
- Substrate: p-Glu-Arg-Thr-Lys-Arg-AMC (Catalog # ES013), 8 mM in deionized water
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: Gemini EM by Molecular Devices) or equivalent
- Dilute rmFurin to 2 μg/mL in Assay Buffer.
- Dilute Substrate to 100 µM in Assay Buffer.
- Load into a black well plate 50 µL of the 2 μg/mL rmFurin and start the reaction by adding 50 µL of 100 µM 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).
- rmFurin: 0.1 µg
- Substrate: 50 µM
Reconstitution Calculator
Background: Furin
Furin is a member of the proprotein convertase (PC) family, which belongs to the subtilisin superfamily of serine proteases. As a cellular protease, Furin processes a variety of proproteins in secretory pathway compartments by cleaving after Arg‑Xaa‑Lys/Arg‑Arg‑like motifs, which usually reside at the end of the pro regions of these proproteins. Examples of the proprotein substrates are growth factors and receptors, extracellular matrix proteins, and other proteases. Furin has an essential role in embryogenesis and homeostasis and is implicated in various pathologies such as cancer, neurodegenerative diseases and anthrax (1, 2). Mouse Furin is a 793 amino acid type I transmembrane protein precursor with a signal peptide (residues 1‑24), a pro region (residues 25‑107), which play a crucial role in the folding, activation and transport of Furin, and a mature chain (residues 108‑793) (2, 3). Mouse Furin displays ~94% homology to the human Furin sequence and >99% homology to the subtilisin‑like catalytic domain (3). The mature chain consists of the subtilisin‑like catalytic domain, a P domain, which is essential for enzyme activity and the modulation of pH and calcium requirements, and a cytoplasmic domain, which controls the localization and sorting of Furin in the trans‑Golgi network/endosomal system (1). The purified recombinant mouse Furin (residues 108‑714) corresponds to the mature enzyme terminated before the transmembrane domain.
- Thomas, G. (2002) Nature Rev. Mol. Cell Biol. 3:753.
- Creemers, J.W. and W.J. Van de Ven. (2004) in Handbook of Proteolytic Enzymes (ed. Barrett, et al.), pp. 1531, Academic Press, San Diego.
- Hatsuzawa, K. et al. (1990) J. Biol. Chem. 265:22075.
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