Recombinant Human Coagulation Factor XIV/Protein C, CF Summary
Met1-Pro461, with a C-terminal 10-His tag
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 Sodium Acetate 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, 150 mM NaCl, 10 mM CaCl2, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
- Assay Buffer: 50 mM Tris, 100 mM NaCl, 0.01% (w/v) Brij-35, pH 8.5
- Recombinant Human Coagulation Factor XIV/Protein C (rhPROC) (Catalog # 3349-SE)
- Bacterial Thermolysin (Thermolysin) (Catalog # 3097-ZN)
- 1,10-Phenanthroline (Sigma, Catalog # 320056), 0.6 M stock in DMSO
- Fluorogenic Peptide 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 100 µg/mL in Activation Buffer with 2.3 µg/mL Thermolysin.
- Incubate for 30 minutes at 37 °C.
- Stop Thermolysin activity by adding 1,10-Phenanthroline to 4 mM.
- Incubate for 15 minutes at room temperature.
- Dilute rhPROC to 4 ng/µL in Assay Buffer.
- Dilute Substrate to 200 µM in Assay Buffer.
- Load into a black well plate 50 µL of 4 ng/µL rhPROC, 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 200 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 (AMC) (Sigma, Catalog # A-9891).
- rhPROC: 0.200 µg
- Substrate: 100 μM
Background: Coagulation Factor XIV/Protein C
Protein C, also known as Coagulation Factor XIV, is a vitamin K-dependent serine protease synthesized in the liver as a single-chain precursor (1). The N‑terminus consists of a signal peptide (amino acid (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 a serine protease domain (aa 212-450). Present in plasma at 3 to 5 mg/L, protein C plays a key role in anticoagulation. Physiologically, the inactive forms of protein C are converted to the active form by thrombin, which releases the activation peptide. The active protein C cleaves factor 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, protein C deficiency is caused by a genetic mutation which affect protein C activity. A severe recessive form may result in a massive thrombosis, which is fatal to the patient. The recombinant human Protein C consists of both the mature chain and the two disulfide-linked chains, which can be activated by treatment with thermolysin.
- Shen, L. and B. Dahlbäck (2004) in Handbook of Proteolytic Enzymes, Barrett, A.J. et al. eds. p. 1673.
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Fluorogenic Peptide Substrates
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