Recombinant Mouse Serpin D1/Heparin Cofactor II Protein, CF

Catalog #: 1265-PI Datasheet / COA / SDS

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1265-PI-010

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Summary Product Datasheets Carrier Free Data Images Reconstitution Calculator Background Related Research Areas

Recombinant Mouse Serpin D1/Heparin Cofactor II 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 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 <1.2 nM, as measured under the described conditions.

Source

Mouse myeloma cell line, NS0-derived mouse Serpin D1/Heparin Cofactor II protein
Glu24-Ser478, with a C-terminal 10-His tag

Accession #

P49182

N-terminal Sequence
Analysis

Glu24

Predicted Molecular Mass

53 kDa

SDS-PAGE

70-80 kDa, reducing conditions

Product Datasheets

1265-PI

Product Datasheet

COA

SDS

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.

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. 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 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 V_max* (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

Reconstitution Calculator

Background: Serpin D1/Heparin Cofactor II

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.

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.

Entrez Gene IDs

3053 (Human); 15160 (Mouse)

Alternate Names

HC2; HC2LS2; HCF2; HCF2clade D (heparin cofactor), member 1; HC-II; Heparin Cofactor II; HLS2HCII; leuserpin 2; LS2; Protease inhibitor leuserpin-2; Serpin D1; serpin peptidase inhibitor, clade D (heparin cofactor), member 1