Recombinant Mouse TFPI Protein, CF

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

Recombinant Mouse TFPI Protein, CF Summary

• Details of Functionality: Measured by its ability to inhibit trypsin cleavage of a fluorogenic peptide substrate, Mca-RPKPVE-Nval-WRK(Dnp)-NH₂ (Catalog # ES002). The IC₅₀ value is <1 nM, as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived mouse TFPI protein
  Leu29-Lys289, with a C-terminal 10-His tag
• Accession #: O54819
• N-terminal Sequence: Leu29
• Protein/Peptide Type: Recombinant Enzymes
• Gene: Tfpi
• 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:
  • Inhibition Activity
• Theoretical MW: 31 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: 41 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-HCl, NaCl and Brij-35.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain.
• Reconstitution Instructions: Reconstitute at 100 μg/mL in sterile 25 mM Tris and 150 mM NaCl, pH 7.5.
• Assay Procedure:
  • Assay Buffer: 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
  • Recombinant Mouse TFPI (rmTFPI) (Catalog # 2975-PI)
  • Trypsin (Sigma, Catalog # T-1426)
  • 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 Trypsin to 0.25 µg/mL in Assay Buffer.
  2. Prepare a curve of rmTFPI (MW: 31,015 Da) in Assay Buffer. Make the following serial dilutions: 1000, 200, 50, 25, 10, 5, 2, 1 and 0.1 nM.
  3. Combine 25 µL of 0.25 µg/mL Trypsin and 25 µL of rmTFPI serial curve dilutions. Include two controls containing 25 µL of Assay Buffer and 25 µL of 0.25 µg/mL Trypsin.
  4. Incubate reaction mixtures at 37 ºC for 15 minutes.
  5. Dilute incubated reaction mixtures with 200 µL of Assay Buffer (1/5 dilution).
  6. Dilute Substrate to 20 µM in Assay Buffer.
  7. In a plate load 50 µL of the diluted incubated reaction mixtures to wells, and start the reaction by adding 50 µL of 20 µM Substrate.
  8. Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively, in kinetic mode for 5 minutes.
  9. Derive the 50% inhibiting concentration (IC₅₀) for rmTFPI by plotting RFU/min (or specific activity) vs. concentration with 4-PL fitting.
  10. The specific activity for Trypsin 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 MCA-Pro-Leu-OH (Bachem, Catalog # M-1975).

  Per Well:
  • Trypsin: 0.00125 µg
  • rmTFPI curve: 50, 10, 2.5, 1.25, 0.5, 0.25, 0.1, 0.05 and 0.005 nM.
  • Substrate: 10 µM

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Mouse TFPI Protein, CF

• convertin
• EPI
• EPITFPI1LACITFI
• Extrinsic pathway inhibitor
• LACI
• Lipoprotein-associated coagulation inhibitor
• TFPI
• tissue factor pathway inhibitor (lipoprotein-associated coagulation inhibitor)
• tissue factor pathway inhibitor

Background

Tissue Factor Pathway Inhibitor (TFPI), also known as lipoprotein-associated coagulation inhibitor (LACI) and extrinsic pathway inhibitor (EPI), is a physiological inhibitor of extrinsic pathway of coagulation and has biological functions of anticoagulation and anti-inflammation (1). It is a secreted protein with a N-terminal acidic region, three Kunitz (K) domains separated by two linker regions, and a C-terminal basic region (2). The first K domain (residues 50 to 100) inhibits coagulation factor VIIa complexed to tissue factor (TF). The second K domain (residues 121 to 171) inhibits factor Xa. The third K domain (residues 225 to 275) binds to heparin (3). The C-terminal basic region may have several functions. For example, it plays an important role in the binding of TFPI to cell surfaces (2). The purified recombinant mouse TFPI ends at residue 289 and does not contain the last 17 residues (residues 290 to 306) in the C-terminal region. It inhibits the activity of Recombinant Human Coagulation Factor VII (Catalog # 2338-SE) in the presence of Recombinant Human Coagulation Factor III/Tissue Factor (Catalog # 2339-PA).

1. Bai, H. et al. (2005) Thromb Haemost. 93:1055.
2. Bajaj, M.S. et al. (2001) Thromb Haemost. 86:959.
3. Mine, S. et al. (2002) Biochemistry 41:78.

Publications for TFPI (2975-PI)(1)

Publication using 2975-PI

• Ma, JK;Su, LD;Feng, LL;Li, JL;Pan, L;Danzeng, Q;Li, Y;Shang, T;Zhan, XL;Chen, SY;Ying, S;Hu, JR;Chen, XQ;Zhang, Q;Liang, T;Lu, XJ; TFPI from erythroblasts drives heme production in central macrophages promoting erythropoiesis in polycythemia Nature communications 2024-05-10 [PMID: 38729948] (Bioassay, In vivo assay, Mouse, Transgenic Mouse)

Bioinformatics

• Gene Symbol: Tfpi
• Uniprot:
  • O54819()