Recombinant Human TFPI Protein, CF

Catalog # Availability Size / Price Qty
2974-PI-010
Recombinant Human TFPI Protein Enzyme Activity
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Citations (3)
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Recombinant Human TFPI Protein, CF Summary

Product Specifications

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.
Activity
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 <0.75 nM, as measured under the described conditions.
Source
Mouse myeloma cell line, NS0-derived human TFPI protein
Asp29-Lys282, with a C-terminal 10-His tag
Accession #
N-terminal Sequence
Analysis
Asp29
Predicted Molecular Mass
31 kDa
SDS-PAGE
41 kDa, reducing conditions

Product Datasheets

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2974-PI

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.

2974-PI

Formulation Lyophilized from a 0.2 μm filtered solution in NaH2PO4 and NaCl.
Reconstitution Reconstitute at 100 μg/mL in sterile 25 mM Tris and 150 mM NaCl, pH 7.5.
Shipping The product is shipped at ambient temperature. 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 reconstitution.

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 Human TFPI1 (rhTFPI1) (Catalog # 2974-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 rhTFPI1 (MW: 30,525 Da) in Assay Buffer. Make the following serial dilutions: 500, 100, 50, 30, 20, 10, 5, 2.5, and 0.833 nM.
  3. Combine equal volumes of 0.25 µg/mL Trypsin and rhTFPI1 serial curve dilutions. Include two controls containing equal volumes of Assay Buffer and 0.25 µg/mL Trypsin.
  4. Incubate reaction mixtures at 37 °C for 15 minutes.
  5. Dilute incubated reaction mixtures 5-fold in Assay Buffer.
  6. Dilute Substrate to 20 µM in Assay Buffer.
  7. In a plate, load 50 µL of the diluted 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 rhTFPI1 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
  • rhTFPI1 curve: 25, 5, 2.5, 1.5, 1.0, 0.5, 0.25, 0.125, and 0.0417 nM
  • Substrate: 10 µM

Data Image

Enzyme Activity Recombinant Human TFPI Protein Enzyme Activity View Larger

Recombinant Human TFPI (Catalog # 2974-PI) inhibits trypsin cleavage of a fluorogenic peptide substrate, Mca-RPKPVE-Nval-WRK(Dnp)-NH2 (Catalog # ES002).

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: TFPI

Human 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 with by two linker regions, and a C-terminal basic region (2). The first K domain (residues 54 to 104) inhibits coagulation factor VIIa complexed to tissue factor (TF). The second K domain (residues 125 to 175) inhibits factor Xa. The third K domain (residues 217 to 267) binds to heparin (3). The C-terminal basic region may have several functions. For example, it plays an important role in binding of TFPI to cell surfaces (2). The purified Recombinant Human TFPI ends at residue 282 and does not contain the last 20 residues (residues 283 to 302) 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).

References
  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.
Long Name
Tissue Factor Pathway Inhibitor
Entrez Gene IDs
7035 (Human); 21788 (Mouse)
Alternate Names
convertin; EPI; EPITFPI1LACITFI; Extrinsic pathway inhibitor; LACI; Lipoprotein-associated coagulation inhibitor; TFPI; tissue factor pathway inhibitor (lipoprotein-associated coagulation inhibitor); tissue factor pathway inhibitor

Citations for Recombinant Human TFPI Protein, CF

R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.

3 Citations: Showing 1 - 3
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  1. Aberrant stromal tissue factor localisation and mycolactone-driven vascular dysfunction, exacerbated by IL-1beta, are linked to fibrin formation in Buruli ulcer lesions
    Authors: LT Hsieh, SJ Dos Santos, BS Hall, J Ogbechi, AD Loglo, FJ Salguero, MT Ruf, G Pluschke, RE Simmonds
    PloS Pathogens, 2022;18(1):e1010280.
    Species: Human
    Sample Types: Cell Culture Supernates
    Applications: ELISA Capture
  2. Tissue factor-expressing tumor cells can bind to immobilized recombinant tissue factor pathway inhibitor under static and shear conditions in vitro.
    Authors: Che S, DeLeonardis C, Shuler M, Stokol T
    PLoS ONE, 2015;10(4):e0123717.
    Species: Human
    Sample Types: Whole Cells
    Applications: Bioassay
  3. Use of an immunoaffinity-mass spectrometry-based approach for the quantification of protein biomarkers from serum samples of lung cancer patients.
    Authors: Nicol GR, Han M, Kim J, Birse CE, Brand E, Nguyen A, Mesri M, FitzHugh W, Kaminker P, Moore PA, Ruben SM, He T
    Mol. Cell Proteomics, 2008;7(10):1974-82.
    Species: N/A
    Sample Types: N/A
    Applications: Standard

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Fluorogenic Peptide Substrates

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