Recombinant Mouse Coagulation Factor Xa Protein, CF Summary
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.
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 MES, 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.
- 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).
- rmFactor Xa: 0.040 μg
- Substrate: 10 μM
Background: Coagulation Factor X
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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Fluorogenic Peptide Substrates
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