Recombinant Human Thrombomodulin/BDCA-3 Protein, CF

Catalog # Availability Size / Price Qty
3947-PA-010
R&D Systems Recombinant Proteins and Enzymes
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Recombinant Human Thrombomodulin/BDCA-3 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 mediate thrombin activation of protein C.
Source
Mouse myeloma cell line, NS0-derived human Thrombomodulin/BDCA-3 protein
Ala19-Ser515 (Ala473Val), with a C-terminal 6-His tag
Accession #
N-terminal Sequence
Analysis
Ala19
Predicted Molecular Mass
53 kDa
SDS-PAGE
80-105 kDa, reducing conditions

Product Datasheets

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3947-PA

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.

3947-PA

Formulation Supplied as a 0.2 μm filtered solution in Tris and NaCl.
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 opening.

Assay Procedure

Materials
  • Activation Buffer: TCNB (0.05 M Tris, 0.01 M CaCl2, 0.15 M NaCl, 0.05 % Brij-35, pH 7.5)
  • Assay Buffer: 0.05 M Tris, 0.1 M NaCl, 0.01% Brij-35, pH 8.5
  • Recombinant Human Coagulation Factor II/Thrombin (rhThrombin) (Catalog # 1473-SE)
  • Recombinant Human Thrombomodulin/BDCA‑3 (rhTHBD) (Catalog #  3947-PA)
  • Recombinant Human Coagulation Factor XIV/Protein C (rhPROC) (Catalog # 3349-SE)
  • Recombinant Human Serpin C1/Antithrombin-III (rhSerpin C1) (Catalog # 1267-PI)
  • Heparin (Sigma-Aldrich, Catalog # H3393), 20 mg/mL in deionized water
  • 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 4 µg/mL in Activation Buffer.
  2. Dilute rhTHBD to 4 µg/mL in Activation Buffer.
  3. Dilute rhPROC to 40 µg/mL in Activation Buffer.
  4. Combine 25 µL of the diluted rhPROC, 12.5 µL of the diluted rhThrombin, and 12.5 µL of the diluted rhTHBD in a microfuge tube. Incubate for one hour at 37 °C to activate rhPROC. As a control combine 25 µL of the diluted rhPROC, 12.5 µL of the diluted rhThrombin, and 12.5 µL of Activation Buffer in a microfuge tube.
  5. Dilute rhSerpin C1 to 20 µg/mL with 32.4 µg/mL heparin in Activation Buffer.
  6. Add and mix 50 µL of the diluted rhSerpin C1/heparin solution to the reaction vials. 
  7. Incubate at room temperature for 30 minutes to stop rhThrombin activity.
  8. Dilute the reaction mixture 1:5 in Assay Buffer.
  9. Dilute Substrate to 200 µM in Assay Buffer.
  10. Load in a black well plate 50 µL of diluted reaction mixture (step 8), and start the reaction by adding 50 µL of 200 µM Substrate.
  11. Read (top read) immediately in kinetic mode for 5 minutes at excitation and emission wavelengths of 380 nm and 460 nm, respectively.
  12. Calculate specific activity:

     Specific Activity (pmol/min/µg) =

Adjusted Vmax* (RFU/min) x Conversion Factor** (pmol/RFU)
amount of enzyme (µg)

     *Adjusted for control containing no rhTHBD
     **Derived using calibration standard 7-Amino, 4-Methyl Coumarin (Sigma-Aldrich, Catalog # A9891).

Per Well:
  • rhPROC: 0.1 μg
  • rhTHBD: 5 ng
  • 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: Thrombomodulin/BDCA-3

Encoded by the THBD gene, Thrombomodulin is also known as CD141 antigen and blood dendritic cell antigen 3 (BDCA-3). The deduced amino acid (aa) sequence of human THBD (Accession # P07204) predicts a signal peptide (aa 1-18) and a mature chain (aa 19‑575) that consists of following domains: C‑type lectin (aa 31-169), EGF-like (aa 241-281, aa 284-324, aa 325-363, aa 365‑405, aa 404‑440, and aa 441‑481), transmembrane (aa 516 to 539) and cytoplasmic (aa 540‑575). The R&D Systems recombinant human THBD consists of aa 19-515, corresponding to the extracellular portion of the type I membrane protein. Predominantly synthesized by vascular endothelial cells, THBD inhibits coagulation and fibrinolysis (1‑3). It functions as a cell surface receptor and an essential cofactor for active thrombin, which in turn activates protein C and thrombin-activatable fibrinolysis inhibitor (TAFI), also known as carboxypeptidase B2 (CPB2). Activated protein C (APC), facilitated by protein S, degrades coagulation factors Va and VIIIa, which are required for thrombin activation. Activated CPB2 cleaves basic C‑terminal aa residues of its substrates, including fibrin, preventing the conversion of plasminogen to plasmin. In addition, THBD gene polymorphisims are associated with human disease and THBD plays a role in thrombosis, stroke, arteriosclerosis, and cancer (4). For example, increased serum levels of THBD, due to protease cleavage, have been associated with smoking, cardiac surgery, atherosclerosis, liver cirrhosis, diabetes mellitus, cerebral and myocardial infarction, and multiple sclerosis (5).

References
  1. Van de Wouwer, M. et al. (2004) Arterioscler. Thromb. Vasc. 24:1374.
  2. Wu, K.K. et al. (2000) Ann. Med. 32:73.
  3. Li, Y.H. et al. (2006) Cardiovasc. Hematol. Agents Med. Chem. 4:183.
  4. Weiler, H. and B.H. Isermann (2003) J. Thromb. Haemost. 1:1515.
  5. Califano, F. et al. (2000) Eur. Rev. Med. Pharmacol. Sci. 4:59.
Entrez Gene IDs
7056 (Human); 21824 (Mouse); 83580 (Rat)
Alternate Names
BDCA3; BDCA-3; CD141 antigen; CD141; Fetomodulin; THBD; THRM; Thrombomodulin; TM

Citations for Recombinant Human Thrombomodulin/BDCA-3 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. Angiopoietin-2 Inhibition of Thrombomodulin-Mediated Anticoagulation-A Novel Mechanism That May Contribute to Hypercoagulation in Critically Ill COVID-19 Patients
    Authors: M Hultström, K Fromell, A Larsson, B Persson, B Nilsson, SE Quaggin, C Betsholtz, R Frithiof, M Lipcsey, M Jeansson
    Biomedicines, 2022;10(6):.
    Species: Human
    Sample Types: Plasma
    Applications: Bioassay
  2. Angiopoietins bind thrombomodulin and inhibit its function as a thrombin cofactor
    Authors: C Daly, X Qian, C Castanaro, E Pasnikowsk, X Jiang, BR Thomson, SE Quaggin, N Papadopoul, Y Wei, JS Rudge, G Thurston, GD Yancopoulo, S Davis
    Sci Rep, 2018;8(1):505.
    Species: Human
    Sample Types: Complex Sample Type
    Applications: Bioassay

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