Recombinant Human Coagulation Factor II/Thrombin Protein, CF

• Reactivity: Hu
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant Human Coagulation Factor II/Thrombin 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 >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 #: P00734
• N-terminal Sequence: Thr328, Ile364 & Val515
• Structure / Form: Active form
• Protein/Peptide Type: Recombinant Enzymes
• Gene: F2
• 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

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 4 kDa (light chain A), 31 kDa (heavy chain B), 14 kDa (C-terminal fragment).
  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: 12 kDa, 21 kDa and 23 kDa, reducing conditions

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 reconstitution.
• Buffer: Lyophilized from a 0.2 μm filtered solution in HEPES and NaCl.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Reconstitution Instructions: Reconstitute at 100 μg/mL in sterile 50 mM HEPES and 580 mM NaCl, pH 7.5.
• Assay Procedure:
  • 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

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Human Coagulation Factor II/Thrombin Protein, CF

• 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

Background

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.

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.

Publications for Coagulation Factor II/Thrombin (1473-SE)(4)

Publications using 1473-SE

• Levengood, MR;Carosino, CM;Zhang, X;Lucas, S;Ortiz, DJ;Westendorf, L;Chin, AP;Martin, AD;Wong, A;Hengel, SM;Sun, H;Zeng, W;Yumul, R;Dominguez, MM;Chen, Y;Zheng, JH;Karlsson, CAB;Trang, VH;Senter, PD;Gardai, SJ; Preclinical development of SGN-CD47M: Protease-activated antibody technology enables selective tumor targeting of the innate immune checkpoint receptor CD47 Molecular cancer therapeutics 2024-10-28 [PMID: 39463068] (Bioassay, N/A)
• 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 Syndecan-4 Protects the Heart From the Profibrotic Effects of Thrombin-Cleaved Osteopontin J Am Heart Assoc, 2020-01-31;9(3):e013518. 2020-01-31 [PMID: 32000579] (Cell Culture, Mouse)
• Mogami , Haruta, Keller , Patrick, Shi , Haolin, Word , R Ann Effect of thrombin on human amnion mesenchymal cells, mouse fetal membranes, and preterm birth. J Biol Chem, 2014-03-20;289(19):13295-307. 2014-03-20 [PMID: 24652285] (In Vivo, Mouse)
• Wu FX, Wang Q, Zhang ZM, Huang S, Yuan WP, Liu JY, Ban KC, Zhao YN Identifying serological biomarkers of hepatocellular carcinoma using surface-enhanced laser desorption/ionization-time-of-flight mass spectroscopy. Cancer Lett., 2009-03-05;279(2):163-70. 2009-03-05 [PMID: 19268439] (ELISA Developmet, Human)

FAQs for Coagulation Factor II/Thrombin (1473-SE). (Showing 1 - 1 of 1 FAQs).

1. Do you have neutralizing antibodies for anti-mouse Thrombin?
   • We currently have 1 neutralizing Thrombin antibody (NBP1-95029), however it is an anti-human antibody and has only been validated in human samples. If you would like to test this antibody in mouse samples, you may be interested in participating in our Innovators Reward Program.

Bioinformatics

• Gene Symbol: F2
• Uniprot:
  • P00734()