>95%, by SDS-PAGE under reducing conditions and visualized by silver stain
<1.0 EU per 1 μg of the protein by the LAL method.
46 kDa. Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
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
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.
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.
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
This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.
Alternate Names for Recombinant Human Coagulation Factor VII Protein, CF
coagulation factor VII (serum prothrombin conversion accelerator)
Coagulation Factor VII
FVII coagulation protein
Serum prothrombin conversion accelerator
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.
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