Recombinant Human Coagulation Factor VII Protein, CF
Recombinant Human Coagulation Factor VII Protein, CF Summary
Product Specifications
Ala39-Pro444, with a C-terminal 10-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.
2338-SE
| Formulation | Supplied as a 0.2 μm filtered solution in Sodium Acetate 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.
|
Assay Procedure
- Processing Buffer: 50 mM Tris, 0.15 M NaCl, 10 mM CaCl2, 0.05% (w/v) Brij-35, pH 7.5
- Assay Buffer: 50 mM Tris, 2.5 mM CaCl2, pH 8.5
- Recombinant Human Coagulation Factor VII (rhF7) (Catalog # 2338-SE)
- Substrate: BOC-Val-Pro-Arg-AMC, (Catalog # ES011), 10 mM stock in DMSO
- Bacterial Thermolysin (Thermolysin) (Catalog # 3097-ZN)
- 1,10-Phenanthroline (Sigma-Aldrich, Catalog # 320050), 0.6 M in DMSO
- Recombinant Human Coagulation Factor III/Tissue Factor (rhTF) (Catalog # 2339-PA)
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Processing
- Incubate rhF7 at 100 µg/mL with Thermolysin at 6.25 µg/mL in Processing Buffer for 20 minutes at 37 °C.
- Stop Thermolysin activity by adding 1,10-Phenanthroline, diluted in Processing Buffer, to a final concentration of 10 mM.
- Incubation at room temperature for 5 minutes.
- Activation
- Dilute the above processed rhF7 to 32 µg/mL with Assay Buffer.
- Dilute rhTF to 35.5 µg/mL with Assay Buffer.
- Combine equal volumes of rhF7 (now at 16 µg/mL) and rhTF (now at 17.75 µg/mL) and incubate for 5 minutes at 37 °C.
- Activity Assay
- Dilute substrate to 200 µM in Assay Buffer.
- Transfer 50 µL of activated rhF7 into wells of a black well plate.
- Start the reaction by adding 50 μL of 200 µM substrate per well.
- Read (top read) in kinetic mode for 5 minutes at excitation and emission wavelengths of 380 nm and 460 nm, respectively.
- Calculate specific activity:
|
Specific Activity (pmoles/min/µg) = |
Adjusted Vmax* (RFU/min) x Conversion Factor** (pmole/RFU) |
| amount of enzyme (µg) |
*Adjusted for Substrate Blank
**Derived using calibration standard 7-Amino, 4-Methyl Coumarin (Sigma-Aldrich, Catalog # A9891).
- rhF7: 0.8 µg
- rhTF: 0.89 µg
- Substrate: 100 µM
Reconstitution Calculator
Background: Coagulation Factor VII
Coagulation Factors VII and VIIa refer to the pro and active forms of the same protease, respectively (1). Factor VII is synthesized in the liver and circulates in the plasma where it binds to tissue factor (TF), an integral membrane protein found in a variety of cell types. Upon binding of TF, Factor VII is rapidly converted into 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). The cleavage and activation of Coagulation Factors VII, IX and X by VIIa:TF is phospholipid-dependent whereas the cleavage of small peptide substrates is not (1). The predominant splicing variant of Factor VII in normal liver corresponds to the 444 amino acid precursor (3, 4). After a signal peptide (residues 1 to 38), the mature chain can be further processed into the light chain (residues 39 to 190) and the heavy chain (residues 191 to 444). The purified Recombinant Human Factor VII corresponds to the mature chain, which can be processed and activated by treatment with thermolysin and binding with Recombinant Human Coagulation Factor III/Tissue Factor (Catalog # 2339-PA) under the conditions described above.
- Morrissey, J.H. (2004) in Handbook of Proteolytic Enzymes, Barrett, A.J. et al. eds. p. 1659.
- Versteeg, H.H. et al. (2003) Carcinogenesis 24:1009.
- Hagen, F.S, et al. (1986) Proc. Natl. Acad. Sci. USA 83:2412.
- O’Hara, P.J. et al. (1987) Proc. Natl. Acad. Sci. USA 84:5158.
Citations for Recombinant Human Coagulation Factor VII 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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Citations: Showing 1 - 2
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Transcription Factor NFAT5 Promotes Migration and Invasion of Rheumatoid Synoviocytes via Coagulation Factor III and CCL2
Authors: S Lee, JS Kong, S You, HM Kwon, SA Yoo, CS Cho, WU Kim
J. Immunol., 2018-05-23;0(0):.
Species: Human
Sample Types: Whole Cells
Applications: Bioassay -
AAV-mediated gene transfer in the perinatal period results in expression of FVII at levels that protect against fatal spontaneous hemorrhage.
Authors: Binny C, McIntosh J, Della Peruta M, Kymalainen H, Tuddenham EG, Buckley SM, Waddington SN, McVey JH, Spence Y, Morton CL, Thrasher AJ, Gray JT, Castellino FJ, Tarantal AF, Davidoff AM, Nathwani AC
Blood, 2011-12-01;119(4):957-66.
Species: Human
Sample Types: N/A, Plasma
Applications: ELISA (Capture), ELISA (Standard)
FAQs
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Is the addition of Coagulation Factor III/Tissue Factor necessary for the activity of Coagulation Factor VII?
Our testing has shown minimal detectable kinetic activity of Coagulation Factor VII following cleavage with Thermolysin in the absence of Tissue Factor.
Upon binding with Tissue Factor (TF), Coagulation Factor VII is rapidly converted into Coagulation Factor VIIa. The resulting 1:1 complex of VIIa and TF initiates the coagulation pathway. This complex also has important coagulation-independent functions such as angiogenesis.
Reviews for Recombinant Human Coagulation Factor VII Protein, CF
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