Recombinant Human Activated Coagulation Factor Xa, ACFP

R&D Systems | Catalog # ACFP1063

R&D Systems
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Key Product Details

Source

Sf 9 (baculovirus)

Accession Number

P00742

Structure / Form

Active Form. Recombinant Human Coagulation Factor Xa Animal Component Free is prone to proteolytic cleavage at C-terminus. The predominant form of the purified protein lacks the His tag.

Applications

Enzyme Activity
View all Coagulation Factor X Proteins and Enzymes »

Product Specifications

Source

Spodoptera frugiperda, Sf 9 (baculovirus)-derived human Coagulation Factor X protein
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).

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.

N-terminal Sequence Analysis

Phe80, Tyr84, & Ile235

Predicted Molecular Mass

16.6 kDa & 30 kDa

SDS-PAGE

12-15 kDa and 30-36 kDa, reducing conditions

Activity

Measured by its ability to cleave the fluorogenic peptide substrate, Mca-RPKPVE-Nval-WRK(Dnp)-NH2 (Catalog # ES002).
The specific activity is >800 pmol/min/μg, as measured under the described conditions.

Formulation, Preparation, and Storage

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

Calculators

The reconstitution calculator allows you to quickly calculate the volume of a reagent to reconstitute your vial. Simply enter the mass of reagent and the target concentration and the calculator will determine the rest.

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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.

Alternate Names

Cf10, F10, FX, FXa, Prothrombinase, Stuart Factor

Entrez Gene IDs

2159 (Human); 14058 (Mouse); 29243 (Rat)

Gene Symbol

F10

UniProt

P00742

Additional Coagulation Factor X Products

Product Documents for Recombinant Human Activated Coagulation Factor Xa, ACFP

Certificate of Analysis

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Note: Certificate of Analysis not available for kit components.

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Product Specific Notices for Recombinant Human Activated Coagulation Factor Xa, ACFP

For research use or further manufacturing only

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Protocols

View specific protocols for Recombinant Human Activated Coagulation Factor Xa, ACFP (ACFP1063):

Materials
  • 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
  1. Dilute rhFactor Xa to 0.4 ng/μL in Assay Buffer.
  2. Dilute Substrate to 20 μM in Assay Buffer.
  3. 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.
  4. Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively, in kinetic mode for 5 minutes.
  5. 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

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