Recombinant Human Activated Coagulation Factor Xa, ACFP
Recombinant Human Activated Coagulation Factor Xa, ACFP Summary
Product Specifications
Met1-Lys488, with a C-terminal 10-His tag
Proform Factor X was expressed, purified, activated and further purified to yield Factor Xa. Produced in an animal component free process (ACFP).
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.
ACFP1063
| Formulation | Lyophilized from a 0.2 μm filtered solution in MES, NaCl and CaCl2. |
| Reconstitution | Reconstitute at 100 μg/mL in sterile 25 mM MES, 150 mM NaCl and 5 mM CaCl2, pH 6.0 |
| 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: 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% Brij-35, pH 7.5 (TCNB)
- Recombinant Human Coagulation Factor Xa Animal Component Free (rhFactor Xa) (Catalog # ACFP1063)
- Substrate: Fluorogenic Peptide Substrate II, Mca-Arg-Pro-Lys-Pro-Val-Glu-Nval-Trp-Arg-Lys(Dnp)-NH2 (Catalog # ES002)
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Dilute rhFactor Xa to 0.4 ng/μL in Assay Buffer.
- Dilute Substrate to 20 μM in Assay Buffer.
- Load 50 μL of the 0.4 ng/μL rhFactor Xa into plate and start the reaction by adding 50 μL of 20 μM Substrate. Include a Substrate Blank containing 50 μL Assay Buffer and 50 μL of 20 μM Substrate without any rhFactor Xa.
- Read at excitation and emission wavelengths of 320 nm and 405 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 MCA-Pro-Leu-OH (Bachem, Catalog # M-1975).
- rhFactor Xa: 0.02 μg
- Substrate: 10 μM
Reconstitution Calculator
Background: Coagulation Factor X
As the only known physiological activator of thrombin, Factor X is a vitamin K-dependent plasma protease that plays a pivotal role in blood coagulation. Human Factor X is initially synthesized in the liver as a single-chain precursor of 488 amino acid residues with a signal peptide and a pro region (residues 1‑40). Both the intrinsic and extrinsic pathways activate Factor X to Xa, which consists of light (residues 41‑179) and heavy (residues 235‑488) chains linked by a disulfide bond. The light chain contains a Gla and two EGF‑like domains and the heavy chain corresponds to the serine protease domain. The full-length human Factor X was expressed and the pro enzyme was purified and activated. The active protease (rhFactor Xa) was further purified and analyzed for its activity towards either peptides or proteins containing a Xa cleavage site. In addition to the activity described in the Activity Assay Protocol, rhFactor Xa also can be used to cleave fusion proteins containing a Factor Xa cleavage site. The conditions for the optimal cleavage of a particular fusion protein, such as the molar ratio between rhFXa and the protein and time and temperature of incubation, are protein-dependent and need to be individually determined.
Manufacturing Specifications
Animal Component-Free Process (ACFP) Manufacturing ConditionsR&D Systems Animal Component-Free Process (ACFP) recombinant proteins are expressed in an animal-free certified Sf 9 insect cell line using dedicated animal-free raw materials and labware. Production and purification procedures use equipment and media that are confirmed animal-free but performed outside our dedicated animal-free laboratories. Every stage of the manufacturing process follows R&D Systems' stringent Standard Operating Procedures (SOPs). The certified Sf 9 insect cell bank has undergone extensive testing to certify the lack of cytopathogens by screening for various viruses, Mycoplasma, and Spiroplasmas using both in vitro and in vivo testing methods. For ex vivo research or bioproduction, additional documentation can be provided.
Please read our complete ACFP Statement
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