Recombinant Mouse TFPI Protein, CF Summary
Leu29-Lys289, with a C-terminal 10-His tag
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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.
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.
|Formulation||Lyophilized from a 0.2 μm filtered solution in Tris-HCl, NaCl and Brij-35.|
|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.
- 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
- Dilute Trypsin to 0.25 µg/mL in Assay Buffer.
- 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.
- 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.
- Incubate reaction mixtures at 37 ºC for 15 minutes.
- Dilute incubated reaction mixtures with 200 µL of Assay Buffer (1/5 dilution).
- Dilute Substrate to 20 µM in Assay Buffer.
- 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.
- Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively, in kinetic mode for 5 minutes.
- Derive the 50% inhibiting concentration (IC50) for rmTFPI by plotting RFU/min (or specific activity) vs. concentration with 4-PL fitting.
- 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).
- 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
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).
- Bai, H. et al. (2005) Thromb Haemost. 93:1055.
- Bajaj, M.S. et al. (2001) Thromb Haemost. 86:959.
- Mine, S. et al. (2002) Biochemistry 41:78.
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Fluorogenic Peptide Substrates
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