Recombinant Mouse Coagulation Factor III/Tissue Factor, CF
Recombinant Mouse Coagulation Factor III/Tissue Factor, CF Summary
Product Specifications
Ala29-Glu251, with a C-terminal 6-His tag
Analysis
Product Datasheets
Carrier Free
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.
3178-PA
| Formulation | Lyophilized from a 0.2 μm filtered solution in Tris, NaCl and Brij-35. |
| Reconstitution | Reconstitute at 200 μg/mL in sterile 50 mM Tris, pH 9.0. |
| 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 Procedure
- Buffer A: 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
- Buffer B: 50 mM Tris, pH 9.0
- Recombinant Mouse Coagulation Factor III/Tissue Factor (rmTF) (Catalog # 3178-PA)
- Recombinant Mouse Coagulation Factor VII (rmF7) (Catalog # 3305-SE)
- Bacterial Thermolysin (Thermolysin) (Catalog # 3097-ZN)
- 1,10 Phenanthroline (Sigma, Catalog # 320056), 600 mM Stock in DMSO
- Substrate: t-Butyloxycarbonyl-Val-Pro-Arg-7-amido-4-methylcoumarin (Catalog # ES011)
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Activate rmF7 at 100 µg/mL in Buffer A containing 10 µg/mL Thermolysin.
- Incubate for 30 minutes at 37 °C.
- Stop reaction by adding 1,10 phenanthroline to a final concentration of 10 mM in Buffer A.
- Incubate for 5 minutes at 37 °C.
- Prepare a curve of rmTF in Buffer B. Make the following serial dilutions: 90, 45, 22.5, 11.3, 5.62, 2.81, 1.41, and 0.703 µg/mL.
- Dilute Thermolysin-activated rmF7 to 32 µg/mL in Buffer B.
- Load in a plate 25 µL of each of the rmTF curve dilutions from step 5. Include two blank controls containing 25 µL of Buffer B and no rmTF.
- Add 25 µL of 32 µg/mL Thermolysin-activated rmF7 to the wells containing rmTF as well as the control wells.
Incubate the reaction in the microplate for 5 minutes at 37 °C. - Dilute Substrate to 200 µM in Buffer B.
- Add 50 µL of 200 µM Substrate to each well containing rmF7 to start the reaction.
- Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in kinetic mode for 5 minutes.
- Derive the 50% activating concentration (AC50) for rmTF by plotting RFU/min (or specific activity) vs. concentration with 4-PL fitting.
- The specific activity for rmF7 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:- rmF7: 0.8 µg
- rmTF: 22.5, 11.3, 5.63, 2.81, 1.41, 0.703, 0.353, and 0.175 µg/mL
- Substrate: 100 µM
Reconstitution Calculator
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 294 amino acid precursor, mouse TF consists of a signal peptide (residues 1 to 28) and the mature chain (residues 29 to 294). As a type I membrane protein, it contains a transmembrane region (residues 252 to 274) and a cytoplasmic tail (residues 275 to 294) (3, 4). The purified rmTF corresponds to the ectodomain (residues 29 to 251) and is potent in activating thermolysin-processed Recombinant Mouse Coagulation Factor VII (Catalog # 3305-SE) under the conditions described in the Activity Assay Protocol.
- Morrissey, J.H. (2004) in Handbook of Proteolytic Enzymes. Barrett, A.J. et al. (ed) Academic Press, San Diego, p. 1659.
- Versteeg, H.H. et al. (2003) Carcinogenesis 24:1009.
- Ranganathan, G. et al. (1991) J. Biol. Chem. 266:496.
- Hartzell, S. (1989) Mol. Cell. Biol. 9:2567.
Citations for Recombinant Mouse 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.
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Citations: Showing 1 - 2
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Rac1 regulates sepsis-induced formation of platelet-derived microparticles and thrombin generation
Authors: Y Wang, L Luo, M Mörgelin, H Thorlacius
Biochem. Biophys. Res. Commun., 2017-04-29;0(0):.
Species: Mouse
Sample Types: Complex Sample Type
Applications: Bioassay -
Targeting tumor endothelial marker 8 in the tumor vasculature of colorectal carcinomas in mice.
Authors: Fernando S, Fletcher BS
Cancer Res., 2009-06-15;69(12):5126-32.
Species: Mouse
Sample Types: Buffer
Applications: Enzyme Assay
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Reason for Rating: Tested the effect of TF on EC proliferation (red) in mice in vivo.
