Recombinant Mouse Coagulation Factor VII Protein, CF Summary
Ala42-Leu446, with a C-terminal 10-His tag
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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 and NaCl.|
|Reconstitution||Reconstitute at 200 μg/mL in sterile 50 mM Tris, 10 mM CaCl2, 150 mM NaCl and 0.05% Brij-35 (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.
- 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, pH 9.0
- Recombinant Mouse Coagulation Factor VII (rmFactor VII) (Catalog # 3305-SE)
- Bacterial Thermolysin (Thermolysin) (Catalog # 3097-ZN)
- 1,10-Phenanthroline (Sigma, Catalog # 320056)
- Recombinant Mouse Coagulation Factor III/Tissue Factor (rmTF) (Catalog # 3178-PA)
- Substrate: Boc-Val-Pro-Arg-AMC (Catalog # ES011), 10 mM stock in DMSO
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Activate rmFactor VII at 100 µg/mL with 10 µg/mL Thermolysin in Activation Buffer.
- Incubate at 37 °C for 30 minutes.
- After incubation, stop reaction with 1,10-Phenanthroline at a final concentration of 10 mM in Activation Buffer.
- Incubate reaction mixtures at 37 °C for 5 minutes. The enzyme concentration is now at 75 µg/mL.
- Dilute rmTF to 15.3 µg/mL in Assay Buffer.
- In a plate, load 13.3 µL of 75 µg/mL activated rmFactor VII followed by adding 36.7 µL of 15.3 µg/mL rmTF.
- Incubate plate at 37 °C for 5 minutes.
- Dilute Substrate to 200 µM in Assay Buffer.
- After plate incubation, start the reaction by adding 50 µL of 200 µM of Substrate to wells.
- Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively in kinetic mode for 5 minutes.
- Calculate specific activity:
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:
- rmFactor VII: 1 µg
- rmTF: 0.56 µg
- Substrate: 100 µM
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 deduced amino acid sequence of mouse factor VII predicts a signal peptide (residues 1 to 24), propeptide (residues 25 to 41), and the mature chain that can be further processed into the light chain (residues 42 to 193) and the heavy chain (residues 194 to 446). The purified recombinant mouse F7 corresponds to the mature chain, which can be processed and activated by treatment with thermolysin and binding with recombinant mouse Tissue Factor (Catalog # 3178-PA) under the conditions described in the Activity Assay Protocol.
- Morrissey, J.H. (2004) in Handbook of Proteolytic Enzymes, Barrett, A.J. et al. (eds. ), Academic Press, San Diego, p. 1659.
- Versteeg, H.H. et al. (2003) Carcinogenesis 24:1009.
Citation for Recombinant Mouse 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.
1 Citation: Showing 1 - 1
Targeting tumor endothelial marker 8 in the tumor vasculature of colorectal carcinomas in mice.
Authors: Fernando S, Fletcher BS
Cancer Res., 2009;69(12):5126-32.
Sample Types: Buffer
Applications: Enzyme Assay
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.
Fluorogenic Peptide Substrates
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