Recombinant Mouse Coagulation Factor Xa Protein, CF

Name: Recombinant Mouse Coagulation Factor Xa Protein, CF
Brand: R&D Systems
SKU: 6724-SE
Rating: 5 (1 reviews)

Catalog #: 6724-SE 1 Review Datasheet / COA / SDS

Catalog #	Availability	Size / Price	Qty
6724-SE-010

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Summary Product Datasheets Carrier Free Data Images Reconstitution Calculator Background Related Research Areas

Recombinant Mouse Coagulation Factor Xa Protein, CF Summary

Product Specifications

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 #

O88947

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

6724-SE

Product Datasheet

COA

SDS

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

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 V_max* (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

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