Recombinant Human TFPI-2 Protein, CF

Catalog #: 2545-PI
2 Citations Datasheet / COA / SDS

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2545-PI-010

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Summary Product Datasheets Carrier Free Data Images Reconstitution Calculator Background Related Research Areas

Recombinant Human TFPI-2 Protein, CF Summary

Product Specifications

Purity

>95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.

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 <5 nM, as measured under the described conditions.

Source

Mouse myeloma cell line, NS0-derived human TFPI-2 protein
Asp23-Lys213, with a C-terminal 10-His tag

Accession #

P48307

N-terminal Sequence
Analysis

Asp23

Predicted Molecular Mass

23 kDa

SDS-PAGE

28-35 kDa, reducing conditions

Product Datasheets

2545-PI

Product Datasheet

COA

SDS

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.

2545-PI

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. 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 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 V_max* (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

Reconstitution Calculator

Background: TFPI-2

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).

References

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.

Long Name

Tissue Factor Pathway Inhibitor 2

Entrez Gene IDs

7980 (Human)

Alternate Names

Placental protein 5; PP5; PP5FLJ21164; REF-1; TFPI2; TFPI-2; TFPI-2REF1; tissue factor pathway inhibitor 2

Related Research Areas

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.

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