Recombinant Human Coagulation Factor II/Thrombin Protein, CF

Catalog # Availability Size / Price Qty
1473-SE-010
Product Details
Citations (2)
FAQs
Supplemental Products
Reviews (1)

Recombinant Human Coagulation Factor II/Thrombin Protein, CF Summary

Product Specifications

Purity
>95%, by SDS-PAGE under reducing conditions and visualized by silver stain
Endotoxin Level
<1.0 EU per 1 μg of the protein by the LAL method.
Activity
Measured by its ability to cleave the fluorogenic peptide substrate Boc-VPR-AMC (Catalog # ES011). The specific activity is >20,000 pmol/min/µg, as measured under the described conditions.
Source
Mouse myeloma cell line, NS0-derived human Coagulation Factor II/Thrombin protein
Met1-Glu622 with a C-terminal 10-His tag
The proenzyme was purified, activated and further purified.
Accession #
N-terminal Sequence
Analysis
Thr328, Ile364 & Val515
Structure / Form
Active form
Predicted Molecular Mass
4 kDa (light chain A), 31 kDa (heavy chain B), 14 kDa (C-terminal fragment)
SDS-PAGE
12 kDa, 21 kDa and 23 kDa, reducing conditions

Product Datasheets

Carrier Free

What does CF mean?

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.

What formulation is right for me?

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.

1473-SE

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.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after reconstitution.

Assay Procedure

Materials
  • 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
  1. Dilute rhThrombin to 0.04 µg/mL in Assay Buffer.
  2. Dilute Substrate to 200 µM in Assay Buffer.
  3. 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.
  4. Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in kinetic mode for 5 minutes.
  5. 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
Reconstitution Calculator

Reconstitution Calculator

The reconstitution calculator allows you to quickly calculate the volume of a reagent to reconstitute your vial. Simply enter the mass of reagent and the target concentration and the calculator will determine the rest.

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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.

References
  1. Degen, S.J. and E.W. Davie (1987) Biochemistry 26:6165.
  2. Coughlin, S.R. (2000) Nature 407:258.
  3. Xi, G. et al. (2003) J. Neurochem. 84:3.
  4. Rydel, T.J. et al. (1994) J. Biol. Chem. 269:22000.
Entrez Gene IDs
2147 (Human); 14061 (Mouse)
Alternate Names
coagulation factor II (thrombin) receptor-like 2; Coagulation factor II receptor-like 2 (protease-actovated receptor 3); Coagulation factor II receptor-like 2; Coagulation Factor II; F2; PAR-3; PAR3proteinase-activated receptor 3; protease-activated receptor 3; proteinase-activated receptor-3; PT; serine protease; Thrombin receptor-like 2; Thrombin

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.

2 Citations: Showing 1 - 2
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  1. 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.
  2. 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.
    Species: Human
    Sample Types: Serum
    Applications: ELISA Developmet

FAQs

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Fluorogenic Peptide Substrates

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Recombinant Human Coagulation Factor II/Thrombin Protein, CF
By Anonymous on 03/30/2019
Application: Used thrombin for the growth of mamospheres.