Recombinant Human Coagulation Factor II/Thrombin Protein, CF Summary
Met1-Glu622 with a C-terminal 10-His tag
The proenzyme was purified, activated and further purified.
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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 HEPES and NaCl.|
|Reconstitution||Reconstitute at 100 μg/mL in sterile 50 mM HEPES and 580 mM NaCl, pH 7.5.|
|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 Buffer: 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
- Recombinant Human Coagulation Factor II/Thrombin (rhThrombin) (Catalog # 1473-SE)
- Substrate: BOC-Val-Pro-Arg-AMC (Catalog # ES011)
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Dilute rhThrombin to 0.04 µg/mL in Assay Buffer.
- Dilute Substrate to 200 µM in Assay Buffer.
- Load 50 µL of 0.04 µg/mL rhThrombin into a plate, and start the reaction by adding 50 µL of 200 µM Substrate. Include a Substrate Blank containing 50 µL of Assay Buffer and 50 µL of Substrate.
- 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:
- rhThrombin: 0.002 µg
- Substrate: 100 µM
Background: Coagulation Factor II/Thrombin
Coagulation Factor II, commonly known as thrombin, is an essential component of the coagulation cascade in which it converts fibrinogen to fibrin, activates factors V, VII, VIII, XIII and forms complexes with protein C and thrombomodulin (1). It also activates platelets and regulates the behavior of additional cells through protease‑activated receptors (PARs) (2). It may have either protective or deleterious functions, depending on the level and location (3). Its activity is regulated by endogenous inhibitors such as anti-thrombin III (serpin C1) or heparin cofactor II (serpin D1). A plasma serine protease, thrombin is synthesized in the liver as a 622 amino acid precursor with a 24 amino acid signal peptide. Cleavage by itself or by similar enzymes converts the proenzyme to three forms designated as alpha ‑, beta ‑ and gamma ‑thrombin. Composed of a disulfide bond-linked dimer of the light chain (A) (residues 328-363) and the heavy chain (B) (residues 364‑622), alpha -thrombin displays the diverse functions as described above. In comparison, the further processed B chains of beta - and gamma -thrombin have no known physiological function, but retain most of the activity towards small synthetic substrates (4). The recombinant human thrombin may be used to process proteins containing the thrombin cleavage site. The conditions for processing different proteins, such as the ratio of protein/thrombin, buffer components, temperature and time of incubation, may vary and should be empirically determined.
- Degen, S.J. and E.W. Davie (1987) Biochemistry 26:6165.
- Coughlin, S.R. (2000) Nature 407:258.
- Xi, G. et al. (2003) J. Neurochem. 84:3.
- Rydel, T.J. et al. (1994) J. Biol. Chem. 269:22000.
Citations for Recombinant Human Coagulation Factor II/Thrombin 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.
Citations: Showing 1 - 3
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Syndecan-4 Protects the Heart From the Profibrotic Effects of Thrombin-Cleaved Osteopontin
Authors: KM Herum, A Romaine, A Wang, AO Melleby, ME Strand, J Pacheco, B Braathen, P Dunér, T Tønnessen, IG Lunde, I Sjaastad, C Brakebusch, AD McCulloch, MF Gomez, CR Carlson, G Christense
J Am Heart Assoc, 2020;9(3):e013518.
Sample Types: Whole Cells
Applications: Cell Culture
Effect of thrombin on human amnion mesenchymal cells, mouse fetal membranes, and preterm birth.
Authors: Mogami, Haruta, Keller, Patrick, Shi, Haolin, Word, R Ann
J Biol Chem, 2014;289(19):13295-307.
Identifying serological biomarkers of hepatocellular carcinoma using surface-enhanced laser desorption/ionization-time-of-flight mass spectroscopy.
Authors: Wu FX, Wang Q, Zhang ZM, Huang S, Yuan WP, Liu JY, Ban KC, Zhao YN
Cancer Lett., 2009;279(2):163-70.
Sample Types: Serum
Applications: ELISA Developmet
What is the theoretical molecular mass of Recombinant Human Coagulation Factor II/Thrombin Protein (catalog 1473-SE)?
The active form of thrombin consists of the light alpha chain and the heavy beta chain. With this understanding, the theoretical molecular mass of our product is ~35 kDa.
What is the C-terminal fragment mentioned in the Product Description?
The 14 kDa C-terminal fragment is a by-product of the activation step and represents a small fraction of the preparation. It has not been removed by any purification steps and appears on SDS-PAGE gels and is the reason we report it.
The product page for Recombinant Human Coagulation Factor II/Thrombin Protein, CF (Catalog # 1473-SE) lists three different molecular weights: light chain A, heavy chain B, and C-terminal fragment. What is the estimated molecular weight of this protein?
The active form of thrombin consists of the light alpha chain and the heavy beta chain. With this understanding, the molecular mass of our 1473-SE protein is ~ 35 kDa. The C-terminal fragment, 14 kDa, is a by-product of the activation step and represents a small fraction of the preparation. It has not been removed by any purification steps and appears on SDS-PAGE gels which is the reason we report it.
Fluorogenic Peptide Substrates
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