Recombinant Human Serpin C1/Antithrombin-III Protein, CF

Catalog # Availability Size / Price Qty
1267-PI-010
Product Details
Citations (4)
FAQs
Supplemental Products
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Recombinant Human Serpin C1/Antithrombin-III Protein, CF Summary

Purity
>95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
Endotoxin Level
<1.0 EU per 1 μg of the protein by the LAL method.
Activity
Measured by its ability to inhibit Recombinant Human Coagulation Factor II/Thrombin (Catalog # 1473-SE) cleavage of a fluorogenic peptide substrate Boc-VPR-AMC (Catalog # ES011). The IC50 value is <5 nM, as measured under the described conditions.
Source
Mouse myeloma cell line, NS0-derived human Serpin C1/Antithrombin-III protein
His33-Lys464, with a C-terminal 10-His tag
Accession #
N-terminal Sequence
Analysis
His33
Predicted Molecular Mass
50 kDa
SDS-PAGE
55-65 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.

1267-PI

Formulation Lyophilized from a 0.2 μm filtered solution in MES and NaCl.
Reconstitution Reconstitute at 100 μg/mL in sterile 25 mM MES, 150 mM NaCl, pH 6.5.
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.
  • 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 Serpin C1/Antithrombin-III (rhSerpin C1) (Catalog # 1267-PI)
  • Recombinant Human Coagulation Factor II/Thrombin (Catalog # 1473-SE)
  • Heparin (Sigma, Catalog # H3393), 20 mg/mL stock in deionized water
  • Substrate: BOC-Val-Pro-Arg-AMC (Catalog # ES011), 10 mM stock in DMSO
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Dilute Thrombin to 1 µg/mL with Heparin at 48.6 µg/mL in Assay Buffer.
  2. Prepare a curve of rhSerpin C1 (MW: 50,378 Da) in Assay Buffer. Make the following serial dilutions: 1000, 500, 250, 125, 62.5, 41.7, 27.8, 13.9, 6.94, and 2.31 nM.
  3. Mix equal volumes of rhSerpin C1 curve dilutions and Thrombin/Heparin mixture. Include a control (in duplicate) containing equal volumes of Assay Buffer and Thrombin/Heparin mixture.
  4. Incubate reaction mixtures at room temperature for 30 minutes.
  5. After incubation, dilute reaction mixtures by 1/5 in Assay Buffer.
  6. Dilute Substrate to 200 µM in Assay Buffer.
  7. In a plate load 50 µL of the diluted reaction mixtures to wells, and start the reaction by adding 50 µL of 200 µM Substrate.
  8. Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in kinetic mode for 5 minutes.
  9. Derive the 50% inhibition concentration (IC50) for rhSerpin C1 by plotting RFU/min (or specific activity) vs. concentration with 4-PL fitting.
  10. Calculate specific activity for Thrombin at each point using the following formula (if needed):

     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:
  • Thrombin: 0.005 µg (1.0 nM)
  • rhSerpin C1:  50, 25, 12.5, 6.25, 3.13, 2.09, 1.39, 0.695, 0.347, and 0.116 nM
  • 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: Serpin C1/Antithrombin-III

Serpin C1 is a member of the Serpin superfamily of the serine protease inhibitors (1). It is the principal plasma Serpin of blood clotting proteases and inhibits thrombin as well as several factors such as Xa (2). Similar to Serpins A5 and D1, its thrombin inhibitory activity is enhanced by heparin. Hereditary and acquired Serpin C1 deficiency is the cause of an increased thrombotic tendency in many cases (3). For example, acquired Serpin C1 deficiency is a common condition in sepsis, after major trauma or surgery (4).

References
  1. Silverman, G.A. et al. (2001) J. Biol. Chem. 276:33293.
  2. Chuang, Y.-J. et al. (2001) Biochemistry 40:6670.
  3. Vinazzer, H. (1999) Semin. Thromb. Hemost. 25:257.
  4. Risberg, B. (1998) Blood Coagul. Fibrinolysis Suppl. 3:S3.
Entrez Gene IDs
462 (Human); 11905 (Mouse)
Alternate Names
Antithrombin-III; AT3antithrombin-III; ATIII; ATIIIantithrombin III; MGC22579; serine (or cysteine) proteinase inhibitor, clade C (antithrombin), member 1; serine-cysteine proteinase inhibitor clade C member 1; Serpin C1; serpin peptidase inhibitor, clade C (antithrombin), member 1

Citations for Recombinant Human Serpin C1/Antithrombin-III 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.

4 Citations: Showing 1 - 4
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  1. Lowering Low-Density Lipoprotein Particles in Plasma Using Dextran Sulphate Co-Precipitates Procoagulant Extracellular Vesicles
    Authors: JW Wang, YN Zhang, SK Sze, SM van de Weg, F Vernooij, AH Schoneveld, SH Tan, HH Versteeg, L Timmers, CSP Lam, DPV de Kleijn
    Int J Mol Sci, 2017;19(1):.
    Applications: Bioassay
  2. Broad spectrum activity of a lectin-like bacterial serine protease family on human leukocytes.
    Authors: Ayala-Lujan, Jorge Lu, Vijayakumar, Vidhya, Gong, Mei, Smith, Rachel, Santiago, Araceli, Ruiz-Perez, Fernando
    PLoS ONE, 2014;9(9):e107920.
    Species: Bacteria
    Sample Types: Protein
    Applications: Enzyme Assay Substrate
  3. Enzymatic properties of human kallikrein-related peptidase 12 (KLK12).
    Authors: Memari&lt;/LastName&gt;&lt;ForeNam N&lt;/Initial, Memari N, Jiang W, Diamandis EP, Luo LY
    Biol. Chem., 2007;388(4):427-35.
    Species: Human
    Sample Types: Recombinant Protein
    Applications: Enzyme Assay
  4. Mannan-binding lectin-associated serine protease 3 cleaves synthetic peptides and insulin-like growth factor-binding protein 5.
    Authors: Cortesio CL, Jiang W
    Arch. Biochem. Biophys., 2006;449(1):164-70.
    Species: N/A
    Sample Types: Protein
    Applications: Enzyme Assay

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