Recombinant Human TFPI-2 Protein, CF
Recombinant Human TFPI-2 Protein, CF Summary
Asp23-Lys213, with a C-terminal 10-His tag
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, 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 Human TFPI‑2 (rhTFPI-2) (Catalog # 2545-PI)
- Trypsin (Sigma, Catalog # T-1426)
- Substrate: MCA-Arg-Pro-Lys-Pro-Val-Glu-NVAL-Trp-Arg-Lys(DNP)-NH2 (Catalog # ES002)
- 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 rhTFPI-2 (MW: 23,197 Da) in Assay Buffer. Make the following serial dilutions: 4000, 800, 400, 200, 100, 50, 25, 12.5, 6.25, and 1.25 nM.
- Combine equal volumes of diluted Trypsin and rhTFPI-2 at each concentration of the curve. Include two controls containing equal volumes of Assay Buffer and diluted Trypsin without any rhTFPI-2.
- Incubate mixtures at 37 °C for 15 minutes.
- Dilute reaction mixtures five fold in Assay Buffer.
- Dilute Substrate to 20 µM in Assay Buffer.
- Load 50 µL of the incubated mixtures in a plate, 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 rhTFPI-2 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).Per Well:
- Trypsin: 0.00125 µg
- rhTFPI-2: 200, 40, 20, 10, 5, 2.5, 1.25, 0.625, 0.3125, and 0.0625 nM.
- Substrate: 10 µM
Human Tissue Factor Pathway Inhibitor 2 (TFPI-2), also known as placental protein 5 (PP5) and retinal pigment epithelial cell factor 1 (REF-1), is a secreted protein with a N-terminal acidic region, three Kunitz (K) domains (residues 36 to 86, 96 to 149 and 158 to 208) separated with by two linker regions, and a C‑terminal basic region (1-3). Expression of TFPI-2 is down-regulated in several cancers, which may contribute to tumor progression in these cancers (4). The purified rhTFPI-2 ends at residue 213 and does not contain the last 22 residues (residues 214 to 235) 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).
- Miyagi, Y. et al. (1994) J. Biochem. 116:939.
- Sprecher, C.A. et al. (1994) Proc. Natl. Acad. Sci. USA 91:3353.
- Tanaka, Y. et al. (2004) Invest. Ophthalmol. Vis. Sci. 45:245.
- Rollin, J. et al. (2005) Br, J. Cancer 92:775.
Citations for Recombinant Human TFPI-2 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.
Citations: Showing 1 - 2
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Clinical Significance of Tissue Factor Pathway Inhibitor 2, a Serum Biomarker Candidate for Ovarian Clear Cell Carcinoma
PLoS ONE, 2016;11(10):e0165609.
Sample Types: N/A
Applications: ELISA (Standard)
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.
Sample Types: N/A
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Fluorogenic Peptide Substrates
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