Recombinant Mouse Coagulation Factor Xa Protein, CF

Catalog # Availability Size / Price Qty
6724-SE-010
Product Details
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Recombinant Mouse Coagulation Factor Xa Protein, CF Summary

Purity
>95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
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, Mca-RPKPVE-Nval-WRK(Dnp)-NH2 (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
Analysis
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.
Predicted Molecular Mass
29 kDa & 10 kDa
SDS-PAGE
30-38 kDa & 13-15 kDa, reducing conditions

Product Datasheets

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.

6724-SE

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.
  • 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, 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
  1. Dilute rmFactor Xa to 0.8 ng/μL in Assay Buffer.
  2. Dilute Substrate to 20 μM in Assay Buffer.
  3. 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.
  4. Read plate at excitation and emission wavelengths of 320 nm and 405 nm, 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).

Per Well:
  • rmFactor Xa: 0.040 μg
  • Substrate: 10 μM
Reconstitution Calculator

Reconstitution Calculator

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

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.

References
  1. Brown, M. A. et al. (2004) in Handbook of Proteolytic Enzymes (eds. Barrett, A. et al.), pp. 1662, Academic Press, San Diego.
Entrez Gene IDs
2159 (Human); 14058 (Mouse); 29243 (Rat)
Alternate Names
Cf10; Coagulation Factor X; EC 3.4.21; EC 3.4.21.6; F10; factor Xa; FX; FXa; Prothrombinase; Stuart Factor; Stuart-Prower factor

FAQs

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Fluorogenic Peptide Substrates

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Recombinant Mouse Coagulation Factor Xa Protein, CF
By Anonymous on 11/03/2016
Application: In vitro bioactivity in cell culture