Babco, Signet, Sternberger Monoclonals, Senetek, Covance

protein

Recombinant Human TFPI (carrier-free)

718908

500 μg

3281 USD

BA

718908

Recombinant Human TFPI (carrier-free)

Tissue factor pathway inhibitor, Extrinsic pathway inhibitor (EPI), Lipoprotein-associated coagulation inhibitor (LACI)

500 μg

3281 USD

Human

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RECOM

Human TFPI Activity AssayHuman 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.Materials1. 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)-NH2Activity Assay Procedures1. 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.;