Recombinant Human Coagulation Factor III/Tissue Factor, CF

R&D Systems | Catalog # 2339-PA

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Key Product Details

  • R&D Systems NS0-derived Recombinant Human Coagulation Factor III/Tissue Factor (2339-PA)
  • Quality control testing to verify active proteins with lot specific assays by in-house scientists
  • All R&D Systems proteins are covered with a 100% guarantee

Source

NS0

Accession Number

P13726

Applications

Enzyme Activity
View all Coagulation Factor III/Tissue Factor Proteins and Enzymes »

Product Specifications

Source

Mouse myeloma cell line, NS0-derived human Coagulation Factor III/Tissue Factor protein
Gly34-Glu251, with a C-terminal 6-His tag

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

Gly34

Predicted Molecular Mass

26 kDa

SDS-PAGE

41 kDa and 43 kDa, reducing conditions

Activity

Measured by its ability to activate Coagulation Factor VII in cleaving a fluorogenic peptide substrate Boc-VPR-AMC (Catalog # ES011).
The AC50 is <4.0 μg/mL, as measured under the described conditions. 

Reviewed Applications

Read 3 reviews rated 5 using 2339-PA in the following applications:

Formulation, Preparation, and Storage

2339-PA
Formulation Supplied as a 0.2 μm filtered solution in Tris and NaCl.
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.

Background: Coagulation Factor III/Tissue Factor

Coagulation Factor III/Tissue Factor (TF), also known as thromboplastin and CD142, is an integral membrane protein found in a variety of cell types. It functions as a protein cofactor/receptor of Coagulation Factor VII, which is synthesized in the liver and circulated in the plasma (1). Upon binding of TF, the inactive factor VII is rapidly converted into activated VIIa. The resulting 1:1 complex of VIIa and TF initiates the coagulation pathway and has also important coagulation‑independent functions such as angiognesis (2). Synthesized as a 295 amino acid precursor, TF consists of a signal peptide (residues 1 to 32) and the mature chain (residues 33 to 295). As a type I membrane protein, it contains a transmembrane region (residues 252 to 274) and a cytoplasmic tail (residues 275 to 295) (3-6). The purified rhTF corresponds to the ectodomain (residues 34 to 251) and is potent in activating thermolysin-processed Recombinant Human Coagulation Factor VII (Catalog # http://www.rndsystems.com/product_results.aspx?k=2338-SE">2338‑SE) under the conditions described in Activity Assay Protocol.

References

  1. Morrissey, J.H. (2004) in Handbook of Proteolytic Enzymes. Barrett, A.J. et al. (ed) San Diego, Academic Press, p. 1659. 
  2. Versteeg, H.H. et al. (2003) Carcinogenesis 24:1009. 
  3. Scarpati, E.M. et al. (1987) Biochemistry 26:5234.
  4. Fisher, K.L. et al. (1987) Thromb. Res. 48:89.
  5. Morrissey, J.H. et al. (1987) Cell 50:129.
  6. Spicer, E.K. (1987) Proc. Natl. Acad. Sci. USA 84:5148.

Alternate Names

CD142, F3, Thromboplastin, Tissue Factor

Entrez Gene IDs

2152 (Human); 14066 (Mouse)

Gene Symbol

F3

UniProt

P13726

Additional Coagulation Factor III/Tissue Factor Products

Product Documents for Recombinant Human Coagulation Factor III/Tissue Factor, CF

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 Coagulation Factor III/Tissue Factor, CF

For research use only

Citations for Recombinant Human Coagulation Factor III/Tissue Factor, CF

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Protocols

View specific protocols for Recombinant Human Coagulation Factor III/Tissue Factor, CF (2339-PA):

Materials
  • Activation Buffer: 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
  • Assay Buffer: 50 mM Tris, 2.5 mM CaCl2, pH 8.5
  • Recombinant Human Coagulation Factor III/Tissue Factor (rhTF) (Catalog # 2339-PA)
  • Recombinant Human Coagulation Factor VII (rhF7) (Catalog # 2338-SE)
  • Bacterial Thermolysin (Thermolysin) (Catalog # 3097-ZN)
  • 1,10 Phenanthroline (Sigma, Catalog # 320056), 600 mM Stock in DMSO
  • Substrate: BOC-Val-Pro-Arg-AMC (R&D Systems, Catalog # ES011), 100 mM in DMSO
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Dilute rhF7 to 100 µg/mL in Activation Buffer containing 6.25 µg/mL Thermolysin.
  2. Incubate at 37 °C for 30 minutes to activate rhF7.
  3. Stop reaction by adding 1,10 phenanthroline to a final concentration of 10 mM in Activation Buffer.
  4. Incubate for 5 minutes at room temperature.
  5. Prepare a dilution curve of rhTF in Assay Buffer. Make serial dilutions of: 71.04, 35.52, 17.76, 8.88, 4.44, 2.22, 1.11, 0.555, 0.278, and 0.139 µg/mL.
  6. Dilute Thermolysin-activated rhF7 to 32 µg/mL in Assay Buffer.
  7. Load 25 µL of each dilution from the dilution curve to a plate. Include two controls containing 25 µL Assay Buffer.
  8. Add 25 µL of 32 µg/mL Thermolysin-activated rhF7 to all the wells.
  9. Incubate at 37 °C for 5 minutes.
  10. Dilute Substrate to 200 µM in Assay Buffer.
  11. Add 50 µL of 200 μM Substrate to all wells.
  12. Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in kinetic mode for 5 minutes.
  13. Derive the 50% activating concentration (AC50) of rhTF by plotting RFU/min (or specific activity) vs. concentration with 4-PL fitting.
  14. The specific activity for rhF7 at each point may be determined using the following formula (if needed):

     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 7-amino, 4-methyl coumarin (Sigma, Catalog # A-9891).

Per Well:

  • rhF7: 0.800 µg
  • rhTF: 17.76, 8.88, 4.44, 2.22, 1.11, 0.556, 0.278, 0.139, 0.0695, and 0.0348 µg/mL
  • Substrate: 100 µM

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