Recombinant Human Serpin D1/Heparin Cofactor II Protein, CF

R&D Systems | Catalog # 3198-PI

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Key Product Details

  • R&D Systems Sf 21 (stably transfected)-derived Recombinant Human Serpin D1/Heparin Cofactor II Protein (3198-PI)
  • Quality control testing to verify active proteins with lot specific assays by in-house scientists
  • All R&D Systems proteins are covered with a 100% guarantee

Source

Sf 21 (stably transfected)

Accession Number

P05546

Applications

Inhibition Activity
View all Serpin D1/Heparin Cofactor II Proteins and Enzymes »

Product Specifications

Source

Spodoptera frugiperda, Sf 21 (stably transfected)-derived human Serpin D1/Heparin Cofactor II protein
Gly20-Ser499, with a C-terminal 10-His tag

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.

N-terminal Sequence Analysis

Gly20

Predicted Molecular Mass

56 kDa

SDS-PAGE

62 kDa, reducing conditions

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 <1.5 nM, as measured under the described conditions.

Formulation, Preparation, and Storage

3198-PI
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.

Background: Serpin D1/Heparin Cofactor II

Serpin D1, also known as Heparin Cofactor II, is a member of the Serpin superfamily of the serine protease inhibitors (1). Similar to Serpins A5 and C1, it inhibits thrombin and this activity is enhanced by heparin. Interestingly, a C-terminal His-tagged recombinant Serpin D1 had enhanced heparin effect, which was maintained in a plasma-based thrombin inhibition assay (2). Congenital Serpin D1 deficiency is a potential risk factor for thrombosis (3). The full‑length human cDNA was expressed and the purified protein corresponded to the secreted form with anti‑thrombin activity.

References

  1. Silverman, G.A. et al. (2001) J. Biol. Chem. 276:33293.
  2. Bauman, S.J. et al. (1999) J. Biol. Chem. 274:34556.
  3. Tollefsen, D.M. (2002) Arch. Pathol. Lab. Med. 126:1394.

Alternate Names

HC2, HCF2, Heparin Cofactor II, LS2

Entrez Gene IDs

3053 (Human); 15160 (Mouse)

Gene Symbol

SERPIND1

UniProt

P05546

Additional Serpin D1/Heparin Cofactor II Products

Product Documents for Recombinant Human Serpin D1/Heparin Cofactor II Protein, CF

Certificate of Analysis

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Product Specific Notices for Recombinant Human Serpin D1/Heparin Cofactor II Protein, CF

For research use only

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Protocols

View specific protocols for Recombinant Human Serpin D1/Heparin Cofactor II Protein, CF (3198-PI):

Materials
  • Assay Buffer: 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% (v/v) Bri-35, pH 7.5 (TCNB)
  • Recombinant Human Serpin D1/Heparin Cofactor II (rhSerpin D1) (Catalog # 3198-PI)
  • Recombinant Human Coagulation Factor II/Thrombin (Thrombin) (Catalog # 1473-SE)
  • Heparin (Sigma, Catalog # H3393), 20 mg/mL in deionized water
  • Substrate: Boc-Val-Pro-Arg-AMC (R&D Systems, Catalog # ES011)
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: Spectramax Gemini EM by Molecular Devices) or equivalent
  1. Dilute Thrombin to 0.4 µg/mL with 48.6 µg/mL Heparin in Assay Buffer.
  2. Prepare a curve of rhSerpin D1 (MW: 56,300 Da) in Assay Buffer. Make the following serial dilutions: 2000, 500, 250, 125, 50, 25, 10, 5, and 1 nM.
  3. Mix equal volumes of the rhSerpin D1 curve dilutions and the diluted Thrombin/Heparin mixture. Include a control containing Assay Buffer and the diluted Thrombin/Heparin mixture.
  4. Incubate mixtures at room temperature for 30 minutes.
  5. After incubation, dilute the mixtures by five fold in Assay Buffer.
  6. Dilute Substrate to 200 µM in Assay Buffer.
  7. Load 50 µL of the diluted incubated mixtures in a plate, 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% inhibiting concentration (IC50) value for rhSerpin D1 by plotting RFU/min (or specific activity) vs. concentration with 4‑PL fitting.
  10. The specific activity for Thrombin at each point may be determined 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: 0.002 µg
  • rhSerpin D1 curve: 100, 25, 12.5, 6.25, 2.5, 1.25, 0.5, 0.25, and 0.05 nM
  • Substrate: 100 µM

FAQs

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Associated Pathways

Blood Coagulation Signaling Pathways Blood Coagulation Signaling Pathway Thumbnail