Human Coagulation Factor II/Thrombin Protein, CF
Human Coagulation Factor II/Thrombin Protein, CF Summary
The human plasma used for the isolation of this product was certified by the supplier to be non-reactive for HIV-1/2 and HBsAg negative at the donor level. Human blood products should always be treated in accordance with universal handling precautions.
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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 MES, NaCl and Brij-35.|
|Reconstitution||Reconstitute at 5 mg/mL in sterile, deionized water.|
|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.
- Assay Buffer: 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
- Human Coagulation Factor II/Thrombin (hThrombin) (Catalog # 2196-SE)
- Bacterial Thermolysin (Thermolysin) (Catalog # 3097-ZN)
- 1,10 Phenanthroline (Sigma, Catalog # 320056), 0.6 M stock in DMSO
- 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 hThrombin to 2 mg/mL in Assay Buffer.
- Dilute Thermolysin to 82 μg/mL in Assay Buffer.
- Mix 40 μL of diluted hThrombin and 40 μL of diluted Thermolysin in microtubes.
- Incubate at 37 °C for 15 minutes.
- After incubation, stop the reaction by adding 80 μL of 20 mM 1,10 Phenanthroline.
- Dilute activated hThrombin to 0.2 μg/mL in Assay Buffer.
- Dilute Substrate to 200 μM in Assay Buffer. Load in a black well plate 50 μL of 0.2 mg/mL of activated hThrombin, and start the reaction by adding 50 μL of 200 μM substrate. As a Substrate Blank combine 50 μL of 200 μM substrate and 50 μL of Assay Buffer.
- 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)|
**Derived using calibration standard 7-amino, 4-Methyl Coumarin (Sigma, Catalog # A-9891). Per Well:
- hThrombin: 0.010 μg
- Substrate: 100 μM
Background: Coagulation Factor II/Thrombin
Coagulation factor II/Thrombin is an essential component of the coagulation cascade in which it converts fibrinogen to fibrin, activates coagulation 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 and a 19 residue pro peptide. The mature chain starting at Ala-44 can be further processed by itself or by similar enzymes into several forms including those 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. Compared to alpha -Thrombin, 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). Human Thrombin purified from the plasma corresponds to the mature chain, which can be activated by treatment with thermolysin. In comparison, the recombinant human Thrombin (Catalog # 1473-SE) is the active enzyme similar to the alpha - and gamma -Thrombin.
- 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.
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