Recombinant Mouse Coagulation Factor III/Tissue Factor, CF

• Reactivity: Mu
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant Mouse Coagulation Factor III/Tissue Factor, CF Summary

• Details of Functionality: Measured by its ability to activate Coagulation Factor VII in cleaving a fluorogenic peptide substrate Boc-VPR-AMC (Catalog # ES011). The AC₅₀ is <5 µg/mL, as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived mouse Coagulation Factor III/Tissue Factor protein
  Ala29-Glu251, with a C-terminal 6-His tag
• Accession #: P20352
• N-terminal Sequence: Ala29
• Protein/Peptide Type: Recombinant Enzymes
• Gene: F3
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 26 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: 38 kDa and 44 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 reconstitution.
• Buffer: Lyophilized from a 0.2 μm filtered solution in Tris, NaCl and Brij-35.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Reconstitution Instructions: Reconstitute at 200 μg/mL in sterile 50 mM Tris, pH 9.0.
• Assay Procedure:
  • Buffer A: 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
  • Buffer B: 50 mM Tris, pH 9.0
  • Recombinant Mouse Coagulation Factor III/Tissue Factor (rmTF) (Catalog # 3178-PA)
  • Recombinant Mouse Coagulation Factor VII (rmF7) (Catalog # 3305-SE)
  • Bacterial Thermolysin (Thermolysin) (Catalog # 3097-ZN)
  • 1,10 Phenanthroline (Sigma, Catalog # 320056), 600 mM Stock in DMSO
  • Substrate: t-Butyloxycarbonyl-Val-Pro-Arg-7-amido-4-methylcoumarin (Catalog # ES011)
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Activate rmF7 at 100 µg/mL in Buffer A containing 10 µg/mL Thermolysin.
  2. Incubate for 30 minutes at 37 °C.
  3. Stop reaction by adding 1,10 phenanthroline to a final concentration of 10 mM in Buffer A.
  4. Incubate for 5 minutes at 37 °C.
  5. Prepare a curve of rmTF in Buffer B. Make the following serial dilutions: 90, 45, 22.5, 11.3, 5.62, 2.81, 1.41, and 0.703 µg/mL.
  6. Dilute Thermolysin-activated rmF7 to 32 µg/mL in Buffer B.
  7. Load in a plate 25 µL of each of the rmTF curve dilutions from step 5. Include two blank controls containing 25 µL of Buffer B and no rmTF.
  8. Add 25 µL of 32 µg/mL Thermolysin-activated rmF7 to the wells containing rmTF as well as the control wells.
     Incubate the reaction in the microplate for 5 minutes at 37 °C.
  9. Dilute Substrate to 200 µM in Buffer B.
  10. Add 50 µL of 200 µM Substrate to each well containing rmF7 to start the reaction.
  11. Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in kinetic mode for 5 minutes.
  12. Derive the 50% activating concentration (AC₅₀) for rmTF by plotting RFU/min (or specific activity) vs. concentration with 4-PL fitting.
  13. The specific activity for rmF7 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:
  • rmF7: 0.8 µg
  • rmTF: 22.5, 11.3, 5.63, 2.81, 1.41, 0.703, 0.353, and 0.175 µg/mL
  • Substrate: 100 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 III/Tissue Factor, CF

• CD142 antigen
• CD142
• coagulation factor III (thromboplastin, tissue factor)
• Coagulation Factor III
• F3
• FLJ17960
• TF
• TFA
• Thromboplastin
• Tissue Factor

Background

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 294 amino acid precursor, mouse TF consists of a signal peptide (residues 1 to 28) and the mature chain (residues 29 to 294). As a type I membrane protein, it contains a transmembrane region (residues 252 to 274) and a cytoplasmic tail (residues 275 to 294) (3, 4). The purified rmTF corresponds to the ectodomain (residues 29 to 251) and is potent in activating thermolysin-processed Recombinant Mouse Coagulation Factor VII (Catalog # 3305-SE) under the conditions described in the Activity Assay Protocol.

1. Morrissey, J.H. (2004) in Handbook of Proteolytic Enzymes. Barrett, A.J. et al. (ed) Academic Press, San Diego, p. 1659. 
2. Versteeg, H.H. et al. (2003) Carcinogenesis 24:1009.
3. Ranganathan, G. et al. (1991) J. Biol. Chem. 266:496.
4. Hartzell, S. (1989) Mol. Cell. Biol. 9:2567.

Publications for Coagulation Factor III/Tissue Factor (3178-PA)(2)

Publications using 3178-PA

• Y Wang, L Luo, M Mörgelin, H Thorlacius Rac1 regulates sepsis-induced formation of platelet-derived microparticles and thrombin generation Biochem. Biophys. Res. Commun., 2017-04-29;0(0):. 2017-04-29 [PMID: 28465231] (Bioassay, Mouse)
• Fernando S, Fletcher BS Targeting tumor endothelial marker 8 in the tumor vasculature of colorectal carcinomas in mice. Cancer Res., 2009-06-15;69(12):5126-32. 2009-06-15 [PMID: 19528090] (Enzyme Assay, Mouse)

FAQs for Coagulation Factor III/Tissue Factor (3178-PA). (Showing 1 - 1 of 1 FAQs).

1. Have you tested any of your anti-tissue factor antibodies against pig? I need one for western blot.
   • Unfortunately, none of our Tissue Factor antibodies have yet been tested with pig samples, so we cannot guarantee that any of them will work in pig. Here is a link to Tissue Factor antibodies that have been validated for use in Western blot. If you are interested in testing any of these antibodies against pig samples, you could become eligible for our Innovator's Reward program.

Bioinformatics

• Gene Symbol: F3
• Uniprot:
  • P20352()