Recombinant Mouse Coagulation Factor Xa Protein, CF Summary
| Details of Functionality |
Measured by its ability to cleave the fluorogenic peptide substrate, Mca-RPKPVE-Nval-WRK(Dnp)-NH 2 (Catalog # ES002). The specific activity is >300 pmol/min/μg, as measured under the described conditions. |
| Source |
Spodoptera frugiperda, Sf 21 (baculovirus)-derived mouse Coagulation Factor X protein
Met1-Asn481, with a C-terminal 10-His tag.
Accession # O88947
Proform Factor X was expressed, purified, activated and further purified to yield Factor Xa. |
| Accession # |
|
| N-terminal Sequence |
Tyr84, Ile232 |
| Structure / Form |
Recombinant Mouse Coagulation Factor Xa is prone to proteolytic cleavage at C-terminus. The predominant form of the purified protein lacks the His tag. |
| Protein/Peptide Type |
Recombinant Enzymes |
| Gene |
F10 |
| Purity |
>95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane |
| Endotoxin Note |
<1.0 EU per 1 μg of the protein by the LAL method. |
Applications/Dilutions
| Dilutions |
|
| Theoretical MW |
29 kDa & 10 kDa.
Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors. |
| SDS-PAGE |
30-38 kDa & 13-15 kDa, reducing conditions |
Packaging, Storage & Formulations
| 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.
|
| Buffer |
Supplied as a 0.2 μm filtered solution in MES, NaCl and CaCl2. |
| Purity |
>95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane |
| Assay Procedure |
- Assay Buffer: 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
- Recombinant Mouse Coagulation Factor Xa (rmFactor Xa) (Catalog # 6724-SE)
- Substrate: MCA-Arg-Pro-Lys-Pro-Val-Glu-NVAL-Trp-Arg-Lys-(DNP)-NH2 (Catalog # ES002), 2 mM stock in DMSO
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Dilute rmFactor Xa to 0.8 ng/μL in Assay Buffer.
- Dilute Substrate to 20 μM in Assay Buffer.
- Load into a plate 50 μL of 0.8 ng/μL rmFactor Xa, and start the reaction by adding 50 μL of 20 μM Substrate. For Substrate Blanks, load 50 μL of Assay Buffer and 50 μL of 20 μM Substrate.
- Read plate at excitation and emission wavelengths of 320 nm and 405 nm, 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). Per Well:
- rmFactor Xa: 0.040 μg
- Substrate: 10 μM
|
Notes
This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.
Alternate Names for Recombinant Mouse Coagulation Factor Xa Protein, CF
Background
Factor X is the only known physiological activator of thrombin and plays a critical role in blood coagulation. Factor X is also involved in cell surface responses including cell activation, gene expression and mitogenesis. Factor X is a serine protease that is synthesized in the liver and released into the bloodstream. Mouse Factor X consists of a single‑chain precursor of 481 amino acid residues that contains a signal peptide (residues 1‑20), pro region (residues 21‑40), light chain (residues 41‑180) and heavy chain (residues 184‑481). The light and heavy chains are 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 mouse Factor X was expressed and purified. Upon activation, the active protease (rmFactor Xa) was further purified and analyzed for activity against a peptide substrate. In addition to the activity described in the Activity Assay Protocol, Factor X can also be used to cleave fusion proteins containing a Factor X cleavage site. The conditions for the optimal cleavage of a particular fusion protein must be determined for each individual fusion protein.
- Brown, M. A. et al. (2004) in Handbook of Proteolytic Enzymes (eds. Barrett, A. et al.), pp. 1662, Academic Press, San Diego.
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