Recombinant Human Active Coagulation Factor XIV/ProteinC, CF

Catalog #: 4998-SE Datasheet / COA / SDS

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4998-SE-010

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Recombinant Human Active Coagulation Factor XIV/ProteinC, CF Summary

Product Specifications

Purity

>95%, by SDS-PAGE under reducing conditions and visualized by silver stain.

Endotoxin Level

<1.0 EU per 1 μg of the protein by the LAL method.

Activity

Measured by its ability to cleave the fluorogenic peptide substrate Boc-VPR-AMC (Catalog # ES011). The specific activity is >275 pmol/min/µg, as measured under the described conditions.

Source

Chinese Hamster Ovary cell line, CHO-derived human Coagulation Factor XIV/Protein C protein
Ala43-Pro461, with a C-terminal 10-His tag
The isolated protein was activated by thrombin/thrombomodulin complex and further purified.

Accession #

P04070

N-terminal Sequence
Analysis

Ala43 (light chain) & Leu212 (heavy chain)

Structure / Form

Disulfide-linked heterodimer

Predicted Molecular Mass

19 kDa (light chain), 29 kDa (heavy chain)

SDS-PAGE

22-25 kDa and 40-50 kDa, reducing conditions

Product Datasheets

4998-SE

Product Datasheet

COA

SDS

Carrier Free

What does CF mean?

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.

What formulation is right for me?

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.

4998-SE

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. 6 months from date of receipt, -20 to -70 °C as supplied. 3 months, -20 to -70 °C under sterile conditions after opening.

Assay Procedure

Materials

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 1 mM in Assay Buffer.
Load into plate 50 µL of 2 ng/µL rhPROC, and start the reaction by adding 50 µL of 1 mM Substrate. As a Substrate Blank combine 50 µL of 1 mM 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 V_max* (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).

Per Well:

rhPROC: 0.1 µg
Substrate: 500 μM

Reconstitution Calculator

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.

References

Shen, L. and Dahlbäck, B. (2004) in Handbook of Proteolytic Enzymes, Barrett, A.J. et al. eds. pp. 1673.

Long Name

Vitamin K-dependent Protein C

Entrez Gene IDs

5624 (Human); 19123 (Mouse); 25268 (Rat)

Alternate Names

Anticoagulant protein C; APC; Autoprothrombin IIA; Blood coagulation factor XIV; Coagulation Factor XIV; EC 3.4.21; EC 3.4.21.69; PC; PROC; PROC1; protein C (inactivator of coagulation factors Va and VIIIa); Protein C; vitamin K-dependent protein C