Recombinant Human Coagulation Factor III/Tissue Factor, CF

Catalog # Availability Size / Price Qty
2339-PA-010
Product Details
Citations (3)
FAQs
Supplemental Products
Reviews (1)

Recombinant Human Coagulation Factor III/Tissue Factor, CF Summary

Purity
>95%, by SDS-PAGE under reducing conditions and visualized by silver stain
Endotoxin Level
<1.0 EU per 1 μg of the protein by the LAL method.
Activity
Measured by its ability to activate Coagulation Factor VII in cleaving a fluorogenic peptide substrate Boc-VPR-AMC (Catalog # ES011). The AC50 is <4.0 μg/mL, as measured under the described conditions. 
Source
Mouse myeloma cell line, NS0-derived human Coagulation Factor III/Tissue Factor protein
Gly34-Glu251, with a C-terminal 6-His tag
Accession #
N-terminal Sequence
Analysis
Gly34
Predicted Molecular Mass
26 kDa
SDS-PAGE
41 kDa and 43 kDa, reducing conditions

Product Datasheets

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.

2339-PA

Formulation Supplied as a 0.2 μm filtered solution in Tris and NaCl.
Shipping The product is shipped with polar packs. 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 opening.

Assay Procedure

Materials
  • Activation Buffer: 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
  • Assay Buffer: 50 mM Tris, 2.5 mM CaCl2, pH 8.5
  • Recombinant Human Coagulation Factor III/Tissue Factor (rhTF) (Catalog # 2339-PA)
  • Recombinant Human Coagulation Factor VII (rhF7) (Catalog # 2338-SE)
  • Bacterial Thermolysin (Thermolysin) (Catalog # 3097-ZN)
  • 1,10 Phenanthroline (Sigma, Catalog # 320056), 600 mM Stock in DMSO
  • Substrate: BOC-Val-Pro-Arg-AMC (R&D Systems, Catalog # ES011), 100 mM in DMSO
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Dilute rhF7 to 100 µg/mL in Activation Buffer containing 6.25 µg/mL Thermolysin.
  2. Incubate at 37 °C for 30 minutes to activate rhF7.
  3. Stop reaction by adding 1,10 phenanthroline to a final concentration of 10 mM in Activation Buffer.
  4. Incubate for 5 minutes at room temperature.
  5. Prepare a dilution curve of rhTF in Assay Buffer. Make serial dilutions of: 71.04, 35.52, 17.76, 8.88, 4.44, 2.22, 1.11, 0.555, 0.278, and 0.139 µg/mL.
  6. Dilute Thermolysin-activated rhF7 to 32 µg/mL in Assay Buffer.
  7. Load 25 µL of each dilution from the dilution curve to a plate. Include two controls containing 25 µL Assay Buffer.
  8. Add 25 µL of 32 µg/mL Thermolysin-activated rhF7 to all the wells.
  9. Incubate at 37 °C for 5 minutes.
  10. Dilute Substrate to 200 µM in Assay Buffer.
  11. Add 50 µL of 200 μM Substrate to all wells.
  12. Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in kinetic mode for 5 minutes.
  13. Derive the 50% activating concentration (AC50) of rhTF by plotting RFU/min (or specific activity) vs. concentration with 4-PL fitting.
  14. The specific activity for rhF7 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 7-amino, 4-methyl coumarin (Sigma, Catalog # A-9891).

Per Well:
  • rhF7: 0.800 µg
  • rhTF: 17.76, 8.88, 4.44, 2.22, 1.11, 0.556, 0.278, 0.139, 0.0695, and 0.0348 µg/mL
  • Substrate: 100 µM
Reconstitution Calculator

Reconstitution Calculator

The reconstitution calculator allows you to quickly calculate the volume of a reagent to reconstitute your vial. Simply enter the mass of reagent and the target concentration and the calculator will determine the rest.

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Background: Coagulation Factor III/Tissue Factor

Coagulation Factor III/Tissue Factor (TF), also known as thromboplastin and CD142, is an integral membrane protein found in a variety of cell types. It functions as a protein cofactor/receptor of Coagulation Factor VII, which is synthesized in the liver and circulated in the plasma (1). Upon binding of TF, the inactive factor VII is rapidly converted into activated VIIa. The resulting 1:1 complex of VIIa and TF initiates the coagulation pathway and has also important coagulation‑independent functions such as angiognesis (2). Synthesized as a 295 amino acid precursor, TF consists of a signal peptide (residues 1 to 32) and the mature chain (residues 33 to 295). As a type I membrane protein, it contains a transmembrane region (residues 252 to 274) and a cytoplasmic tail (residues 275 to 295) (3-6). The purified rhTF corresponds to the ectodomain (residues 34 to 251) and is potent in activating thermolysin-processed Recombinant Human Coagulation Factor VII (Catalog # 2338‑SE) under the conditions described in Activity Assay Protocol.

References
  1. Morrissey, J.H. (2004) in Handbook of Proteolytic Enzymes. Barrett, A.J. et al. (ed) San Diego, Academic Press, p. 1659. 
  2. Versteeg, H.H. et al. (2003) Carcinogenesis 24:1009. 
  3. Scarpati, E.M. et al. (1987) Biochemistry 26:5234.
  4. Fisher, K.L. et al. (1987) Thromb. Res. 48:89.
  5. Morrissey, J.H. et al. (1987) Cell 50:129.
  6. Spicer, E.K. (1987) Proc. Natl. Acad. Sci. USA 84:5148.
Entrez Gene IDs
2152 (Human); 14066 (Mouse)
Alternate Names
CD142 antigen; CD142; coagulation factor III (thromboplastin, tissue factor); Coagulation Factor III; F3; FLJ17960; TF; TFA; Thromboplastin; Tissue Factor

Citations for Recombinant Human Coagulation Factor III/Tissue Factor, 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.

3 Citations: Showing 1 - 3
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  1. Quasi-static acoustic tweezing thromboelastometry
    Authors: RG Holt, D Luo, N Gruver, DB Khismatull
    J. Thromb. Haemost., 2017;0(0):.
    Species: Human
    Sample Types: Whole Blood
    Applications: Bioassay
  2. Performance characteristics of combinations of host biomarkers to identify women with occult placental malaria: a case-control study from Malawi.
    Authors: Conroy AL, Liles WC, Molyneux ME
    PLoS ONE, 2011;6(12):e28540.
    Species: N/A
    Sample Types: N/A
    Applications: ELISA (Standard)
  3. Molecular intercommunication between the complement and coagulation systems.
    Authors: Amara U, Flierl MA, Rittirsch D, Klos A, Chen H, Acker B, Bruckner UB, Nilsson B, Gebhard F, Lambris JD, Huber-Lang M
    J. Immunol., 2010;185(9):5628-36.
    Species: Human
    Sample Types: Recombinant Protein
    Applications: Enzyme Assay

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Recombinant Human Coagulation Factor III/Tissue Factor, CF
By Anonymous on 08/03/2016
Application: Enzymatic activity in vitro