Recombinant Human Coagulation Factor VII Protein, CF

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Product Details

Summary
Reactivity HuSpecies Glossary
Applications Enzyme Activity
Format
Carrier-Free

Order Details

Recombinant Human Coagulation Factor VII Protein, CF Summary

Details of Functionality
Measured by its ability to cleave the fluorogenic peptide substrate Boc-VPR-AMC (Catalog # ES011). The specific activity is >50 pmol/min/µg, as measured under the described conditions.
Source
Mouse myeloma cell line, NS0-derived human Coagulation Factor VII protein
Ala39-Pro444, with a C-terminal 10-His tag
Accession #
N-terminal Sequence
Ala39
Structure / Form
Mature form
Protein/Peptide Type
Recombinant Enzymes
Gene
F7
Purity
>95%, by SDS-PAGE under reducing conditions and visualized by silver stain
Endotoxin Note
<1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

Theoretical MW
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.
SDS-PAGE
60 kDa, reducing conditions
Publications
Read Publications using
2338-SE in the following applications:

Packaging, Storage & Formulations

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.
Buffer
Supplied as a 0.2 μm filtered solution in Sodium Acetate and NaCl.
Purity
>95%, by SDS-PAGE under reducing conditions and visualized by silver stain
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
  1. Processing
    1. Incubate rhF7 at 100 µg/mL with Thermolysin at 6.25 µg/mL in Processing Buffer for 20 minutes at 37 °C.
    2. Stop Thermolysin activity by adding 1,10-Phenanthroline, diluted in Processing Buffer, to a final concentration of 10 mM.
    3. Incubation at room temperature for 5 minutes.
  2. Activation
    1. Dilute the above processed rhF7 to 32 µg/mL with Assay Buffer.
    2. Dilute rhTF to 35.5 µg/mL with Assay Buffer.
    3. 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.
  3. Activity Assay
    1. Dilute substrate to 200 µM in Assay Buffer.
    2. Transfer 50 µL of activated rhF7 into wells of a black well plate.
    3. Start the reaction by adding 50 μL of 200 µM substrate per well.
    4. Read (top read) in kinetic mode for 5 minutes at excitation and emission wavelengths of 380 nm and 460 nm, respectively.
    5. 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).

Per Well:
  • rhF7: 0.8 µg
  • rhTF: 0.89 µg
  • Substrate: 100 µM

Notes

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
  • EC 3.4.21
  • EC 3.4.21.21
  • Eptacog alfa
  • F7
  • FVII coagulation protein
  • proconvertin
  • Serum prothrombin conversion accelerator
  • SPCA

Background

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.

  1. Morrissey, J.H. (2004) in Handbook of Proteolytic Enzymes, Barrett, A.J. et al. eds. p. 1659. 
  2. Versteeg, H.H. et al. (2003) Carcinogenesis 24:1009.
  3. Hagen, F.S, et al. (1986) Proc. Natl. Acad. Sci. USA 83:2412.
  4. O’Hara, P.J. et al. (1987) Proc. Natl. Acad. Sci. USA 84:5158.

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2338-SE
Species: Hu
Applications: Enzyme Activity

Publications for Coagulation Factor VII (2338-SE)(2)

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Bioinformatics

Gene Symbol F7
Uniprot