Recombinant Human Active Coagulation Factor XIV/ProteinC, CF
Recombinant Human Active Coagulation Factor XIV/ProteinC, CF Summary
Ala43-Pro461, with a C-terminal 10-His tag
The isolated protein was activated by thrombin/thrombomodulin complex and further purified.
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, NaCl and CaCl2.|
|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 Tris, 0.15 M NaCl, 10 mM CaCl2, 0.01% (w/v) Brij 35, pH 8.5
- Recombinant Human Active Coagulation Factor XIV/Protein C (rhPROC) (Catalog # 4998-SE)
- Substrate: Boc-Val-Pro-Arg-Amc (Catalog # ES011)
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Dilute rhPROC to 2 ng/µL in Assay Buffer.
- Dilute Substrate to 200 µM in Assay Buffer.
- Load into plate 50 µL of 2 ng/µL rhPROC, and start the reaction by adding 50 µL of 200 μM Substrate. As a Substrate Blank combine 50 µL of 200 μM Substrate and 50 µL of Assay Buffer.
- 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 (AMC) (Sigma, Catalog # A-9891).
- rhPROC: 0.1 µg
- Substrate: 100 μM
Background: Coagulation Factor XIV/Protein C
Protein C (PROC) is a vitamin K-dependent serine protease synthesized in liver as a single-chain precursor (1). The N-terminus consists of a signal peptide (aa 1-32) and a propeptide (aa 33-42). The mature chain (aa 43-461) is converted to two disulfide-linked chains (light: aa 43-199 and heavy: 200‑461) and both forms are inactive. The light chain consists of Gla (gamma-carboxy-glutamate) domain and two EGF-like domains. The heavy chain consists of an activation peptide (aa 200‑211) and serine protease domain (aa 212-450). Present in plasma at 3 to 5 mg/L, PROC plays a key role in anticoagulation. Physiologically, the inactive forms of PROC are converted to the active form by thrombin, which releases the activation peptide. The active PROC cleaves factors VIIIa and Va to inactivate them. This anticoagulation activity can be enhanced by a presence of a cofactor such as Protein S. In hereditary thrombophilia, PROC deficiency is caused by a genetic mutation which affects PROC activity. A severe recessive form may result in massive thrombosis fatal to patient.
- Shen, L. and Dahlbäck, B. (2004) in Handbook of Proteolytic Enzymes, Barrett, A.J. et al. eds. pp. 1673.
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Fluorogenic Peptide Substrates
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