Serpin D1 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). Mouse Serpin D1 is synthesized as a 478 amino acid precursor with a 23 amino acid signal peptide (4). The mature chain (residues 24 to 478) is secreted.
Recombinant Mouse Serpin D1/Heparin Cofactor II Protein, CF
R&D Systems | Catalog # 1265-PI
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Key Product Details
- R&D Systems NS0-derived Recombinant Mouse Serpin D1/Heparin Cofactor II Protein (1265-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
NS0
Accession Number
Applications
Inhibition Activity
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Product Specifications
Source
Mouse myeloma cell line, NS0-derived mouse Serpin D1/Heparin Cofactor II protein
Glu24-Ser478, with a C-terminal 10-His tag
Glu24-Ser478, 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
Glu24
Predicted Molecular Mass
53 kDa
SDS-PAGE
70-80 kDa, reducing conditions
Formulation, Preparation, and Storage
1265-PI
| Formulation | Lyophilized from a 0.2 μm filtered solution in HEPES and NaCl. |
| Reconstitution | Reconstitute at 100 μg/mL in sterile 25 mM HEPES and 150 mM NaCl, pH 7.4. |
| 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.
|
Calculators
Background: Serpin D1/Heparin Cofactor II
References
- Silverman, G.A. et al. (2001) J. Biol. Chem. 276:33293.
- Bauman, S.J. et al. (1999) J. Biol. Chem. 274:34556.
- Tollefsen, D.M. (2002) Arch. Pathol. Lab. Med. 126:1394.
- Zhang, G.S. et al. (1994) Biochemistry 33:3632.
Alternate Names
HC2, HCF2, Heparin Cofactor II, LS2
Gene Symbol
SERPIND1
UniProt
Additional Serpin D1/Heparin Cofactor II Products
Product Documents for Recombinant Mouse Serpin D1/Heparin Cofactor II Protein, CF
Certificate of Analysis
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Note: Certificate of Analysis not available for kit components.
Product Specific Notices for Recombinant Mouse Serpin D1/Heparin Cofactor II Protein, CF
For research use only
Related Research Areas
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Protocols
View specific protocols for Recombinant Mouse Serpin D1/Heparin Cofactor II Protein, CF (1265-PI):
Materials
- Assay Buffer: 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
- Recombinant Mouse Serpin D1/Heparin Cofactor II (rmSerpin D1) (Catalog # 1265-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), 100 mM stock in DMSO
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Dilute Thrombin to 2 µg/mL and Heparin to 0.1 mg/mL each in Assay Buffer.
- Mix equal portions of Thrombin and Heparin for final concentrations of 1 µg/mL and 0.05 mg/mL respectively.
- Prepare a curve of rmSerpin D1 (MW: 53,374 Da) in Assay Buffer. Make the following serial dilutions: 200, 100, 50, 25, 15, 10, 5, 2.5, 1.25 and 0.625 nM.
- Mix equal volumes of the rmSerpin D1 curve dilutions and the Thrombin/Heparin mixture. Include a control (in duplicate) containing Assay Buffer and the Thrombin/Heparin mixture.
- Incubate mixtures at room temperature for 30 minutes.
- After incubation, dilute the mixtures 5 fold in Assay Buffer.
- Dilute Substrate to 200 µM in Assay Buffer.
- Load 50 µL of the diluted incubated mixtures in a plate, and start the reaction by adding 50 µL of 200 µM Substrate.
- Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in kinetic mode for 5 minutes.
- Derive the 50% inhibition concentration (IC50) for rmSerpin D1 by plotting RFU/min (or specific activity) vs. concentration with
4-PL fitting.
- 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: 0.005 µg
- Heparin: 2.5 μg/mL
- rmSerpin D1 curve: 10, 5, 2.5, 1.5, 0.75, 0.5, 0.25, 0.125, 0.0625, and 0.03125 nM
- Substrate: 100 µM
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Associated Pathways
