Recombinant Human Serpin D1/Heparin Cofactor II Protein, CF

Catalog #: 3198-PI Datasheet / COA / SDS

Catalog #	Availability	Size / Price	Qty
3198-PI-010

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

Recombinant Human 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 <1.5 nM, as measured under the described conditions.

Source

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

Accession #

P05546

N-terminal Sequence
Analysis

Gly20

Predicted Molecular Mass

56 kDa

SDS-PAGE

62 kDa, reducing conditions

Product Datasheets

3198-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.

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.

Assay Procedure

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

Dilute Thrombin to 0.4 µg/mL with 48.6 µg/mL Heparin in Assay Buffer.
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.
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.
Incubate mixtures at room temperature for 30 minutes.
After incubation, dilute the mixtures by five 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% inhibiting concentration (IC₅₀) value for rhSerpin 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: 0.002 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

Reconstitution Calculator

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

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.

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