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- Regulatory Status
- RUO
- Other Names
- Tissue factor pathway inhibitor, Extrinsic pathway inhibitor (EPI), Lipoprotein-associated coagulation inhibitor (LACI)
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- Product Citations
- publications
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The activity of hTFPI was measured by its ability to inhibit trypsin from bovine pancreas. The potency of inhibition was measured by monitoring the cleavage of a fluorogenic substrate Mca-RPKPVE-Nval-WRK(Dnp)-NH2 (10 µM) in the presence of the trypsin (10 ng/mL). The IC50 value of hTFPI is less than 0.42 nM (14.0 ng/mL).
Tissue factor pathway inhibitor (TFPI), also known as extrinsic pathway inhibitor (EPI), lipoprotein-associated coagulation inhibitor (LACI), is a plasma proteinase inhibitor which controls the tissue factor (TF)-dependent pathway of blood coagulation. The major site of TFPI production is in endothelial cells, which constitutively express the protein under normal conditions. TFPI is also normally expressed by vascular smooth muscle cells, megakaryocytes/platelets, monocytes, mesangial cells fibroblasts, and cardiomyocytes. In addition, TFPI has been detected in macrophages and T cells in atherosclerotic lesions. TFPI contains three tandem Kunitz-type serine protease inhibitor domains, K1, K2, and K3, followed by a basic C-terminal region. K2 directly binds to and inhibits the active site of FXa K1 binds to and inhibits the active site of FVIIa, thereby inhibiting procoagulant activity produced by the TF–FVIIa catalytic complex. An alternatively transcribed isoform with GPI anchor arises through differential splicing of the TFPI mRNA toward the 5’-end of the coding region. This membrane anchored protein, referred to as TFPI-β, is a smaller size protein without K3 and basic c-terminal domain. Upregulators of TFPI expression in vitro that might be of physiological significance include endotoxin, interleukin-1, tumor necrosis factor-α, platelet-derived growth factor, heparin, basic fibroblast growth factor, and elevated shear stress. TFPI is distributed in three ways in vivo; about 80 to 85% of the total body TFPI is bound to endothelial cell-surface, and 15-20% is circulating in plasma, of which 80% is bound to lipoproteins and the rest is the free form.
Product DetailsProduct Details
- Source
- Human TFPI, amino acids Asp29-Met304 (Accession# NM_006287) with a C-terminal TG-8H-GGQ tag was expressed in HEK 293E cells.
- Molecular Mass
- The 289 amino acid recombinant protein has a predicted molecular mass of approximately 33.4 kD. The non-reduced and DTT-reduced proteins migrate at 50 - 60 kD by SDS-PAGE.
- Purity
- > 95% as determined by Coomassie stained SDS-PAGE.
- Formulation
- 0.22 µm filtered protein solution is in PBS at pH 7.2
- Endotoxin Level
- Less than 1.0 EU per µg of protein as determined by the LAL method.
- Concentration
- 10 and 25 µg sizes are bottled at 200 µg/mL. 100 µg size and larger sizes are lot-specific and bottled at the concentration indicated on the vial. To obtain lot-specific concentration and expiration, please enter the lot number in our Certificate of Analysis online tool.
- Storage & Handling
- Unopened vial can be stored between 2°C and 8°C for up to 2 weeks, at -20°C for up to six months, or at -70°C or colder until the expiration date. For maximum results, quick spin vial prior to opening. The protein can be aliquoted and stored at -20°C or colder. Stock solutions can also be prepared at 50 - 100 µg/mL in appropriate sterile buffer, carrier protein such as 0.2 - 1% BSA or HSA can be added when preparing the stock solution. Aliquots can be stored between 2°C and 8°C for up to one week and stored at -20°C or colder for up to 3 months. Avoid repeated freeze/thaw cycles.
- Activity
- Human TFPI inhibits the cleavage of a fluorogenic peptide substrate Mca-RPKPVE-Nval-WRK(Dnp)-NH2 by bovine trypsin with IC50 value less than 0.42 nM (14.0 ng/mL).
- Application
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Bioassay
- Application Notes
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Human TFPI Activity Assay
Human TFPI activity is measured by its ability to inhibit cleavage of a fluorogenic peptide substrate, Mca-RPKPVE-Nval-WRK(Dnp)-NH2, by Trypsin from Bovine pancreas. The potency of hTFPI is described as an IC50 value.
Materials
1. Recombinant human TFPI (Predicted MW: 33.4 kD)
2. Assay Buffer: pH 7.5, 50 mM Tris, 10 mM CaCl2 0.15 M NaCl, 0.05% Brij-35.
3. Trypsin from Bovine pancreas.
4. Trypsin substrate: Mca-RPKPVE-Nval-WRK(Dnp)-NH2
Activity Assay Procedures
1. Dilute hTFPI in 2560 ng/mL in Assay Buffer.
2. Make serial dilution of hTFPI in Assay Buffer from 2560 ng/mL to 10 ng/mL, and include 0 ng/mL as control.
3. Dilute Trypsin in 0.2 µg/mL in Assay Buffer.
4. Add into a tube 20 µL of each serially-diluted hTFPI including the control without hTFPI, and then add 20 µL of 0.2 µg/mL trypsin solution. Briefly vortex, and then centrifuge the tubes to mix the solutions.
5. Incubate the mixture at room temperature for 30 minutes.
6. Add 160 µL of Assay Buffer into each tube prepared in step 4 after incubation to make diluted assay mixtures.
7. Dilute the substrate in Assay Buffer at 20 µM.
8. Load into a well plate 50 μL of each assay mixture and start the reaction by adding 50 μL of 20 μM Substrate. Include a substrate blank containing 50 μL Assay Buffer and 50 μL of 20 μM Substrate.
9. Read at excitation and emission wavelengths of 320 nm and 405 nm, respectively, in kinetic mode for 5 minutes.
10. Analyze the data with non-linear regression to estimate IC50 value of hTFPI towards bovine trypsin.
Final Assay Conditions Per Well
- Trypsin: 1 ng (10 ng/mL)
- hTFPI: 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0 ng/mL
- Substrate: 10 µM
BioLegend carrier-free recombinant proteins provided in liquid format are shipped on blue-ice. Our comparison testing data indicates that when handled and stored as recommended, the liquid format has equal or better stability and shelf-life compared to commercially available lyophilized proteins after reconstitution. Our liquid proteins are verified in-house to maintain activity after shipping on blue ice and are backed by our 100% satisfaction guarantee. If you have any concerns, contact us at tech@biolegend.com.
Antigen Details
- Structure
- Monomer
- Distribution
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Human serum and plasma, high expression in HUVEC, TIME, and LHCN-M2 cell line and medium expression in CAPAN-2 and Hep-G2 cell line.
- Function
- Regulate blood coagulation.
- Interaction
- FXa, FVIIa.
- Ligand/Receptor
- TF-FVIIa complex, lipoproteins, proteoglycans.
- Molecular Family
- Enzymes and Regulators
- Antigen References
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1. Maroney SA, Mast AE. 2015. J. Thromb. Haemost. 13:S200.
2. Wood JP, et al. 2014. Blood. 123:2934.
3. Crawley JT, Lane DA. 2008. Arterioscler. Thromb. Vasc. Biol. 28:233.
4. Broze GJ Jr., et al. 1994. Blood Coagul. Fibrinolysis 5:551. - Gene ID
- 7035 View all products for this Gene ID
- UniProt
- View information about TFPI on UniProt.org
Related FAQs
- Why choose BioLegend recombinant proteins?
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• Each lot of product is quality-tested for bioactivity as indicated on the data sheet.
• Greater than 95% Purity or higher, tested on every lot of product.
• 100% Satisfaction Guarantee for quality performance, stability, and consistency.
• Ready-to-use liquid format saves time and reduces challenges associated with reconstitution.
• Bulk and customization available. Contact us.
• Learn more about our Recombinant Proteins. - How does the activity of your recombinant proteins compare to competitors?
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We quality control each and every lot of recombinant protein. Not only do we check its bioactivity, but we also compare it against other commercially available recombinant proteins. We make sure each recombinant protein’s activity is at least as good as or better than the competition’s. In order to provide you with the best possible product, we ensure that our testing process is rigorous and thorough. If you’re curious and eager to make the switch to BioLegend recombinants, contact your sales representative today!
- What is the specific activity or ED50 of my recombinant protein?
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The specific activity range of the protein is indicated on the product datasheets. Because the exact activity values on a per unit basis can largely fluctuate depending on a number of factors, including the nature of the assay, cell density, age of cells/passage number, culture media used, and end user technique, the specific activity is best defined as a range and we guarantee the specific activity of all our lots will be within the range indicated on the datasheet. Please note this only applies to recombinants labeled for use in bioassays. ELISA standard recombinant proteins are not recommended for bioassay usage as they are not tested for these applications.
- Have your recombinants been tested for stability?
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Our testing shows that the recombinant proteins are able to withstand room temperature for a week without losing activity. In addition the recombinant proteins were also found to withstand four cycles of freeze and thaw without losing activity.
- Does specific activity of a recombinant protein vary between lots?
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Specific activity will vary for each lot and for the type of experiment that is done to validate it, but all passed lots will have activity within the established ED50 range for the product and we guarantee that our products will have lot-to-lot consistency. Please conduct an experiment-specific validation to find the optimal ED50 for your system.
- How do you convert activity as an ED50 in ng/ml to a specific activity in Units/mg?
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Use formula Specific activity (Units/mg) = 10^6/ ED50 (ng/mL)
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