Recombinant Human Serpin D1/Heparin Cofactor II Catalog Number: 3198-PI DESCRIPTION Source Spodoptera frugiperda, Sf 21 (stably transfected)­derived Gly20­Ser499, with a C­terminal 10­His tag Accession # P05546 N­terminal Sequence Gly20 Analysis Predicted Molecular 56 kDa Mass SPECIFICATIONS SDS­PAGE 62 kDa, reducing conditions Activity Measured by its ability to inhibit Recombinant Human Coagulation Factor II/Thrombin (Catalog # 1473­SE) cleavage of a fluorogenic peptide substrate Boc­VPR­AMC (Catalog # ES011). The IC50 value is <1.5 nM, as measured under the described conditions. Endotoxin Level <1.0 EU per 1 μg of the protein by the LAL method. Purity >95%, by SDS­PAGE under reducing conditions and visualized by silver stain. Formulation Supplied as a 0.2 μm filtered solution in Tris and NaCl. See Certificate of Analysis for details. Activity Assay Protocol Materials l Assay Buffer: 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% (v/v) Bri­35, pH 7.5 (TCNB) l Recombinant Human Serpin D1/Heparin Cofactor II (rhSerpin D1) (Catalog # 3198­PI) l Recombinant Human Coagulation Factor II/Thrombin (Thrombin) (Catalog # 1473­SE) l Heparin (Sigma, Catalog # H3393), 20 mg/mL in deionized water l Substrate: Boc­Val­Pro­Arg­AMC (R&D Systems, Catalog # ES011) l F16 Black Maxisorp Plate (Nunc, Catalog # 475515) l Fluorescent Plate Reader (Model: Spectramax Gemini EM by Molecular Devices) or equivalent Assay 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 4­PL fitting. 10. The specific activity for Thrombin at each point may be determined using the following formula (if needed): Adjusted V * (RFU/min) x Conversion Factor** (pmol/RFU) Specific Activity (pmol/min/µg) = max amount of enzyme (µg) *Adjusted for Substrate Blank **Derived using calibration standard 7­amino, 4­Methyl Coumarin (Sigma, Catalog # A­9891). Final Assay Per Well: Conditions l Thrombin: 0.002 µg l rhSerpin D1 curve: 100, 25, 12.5, 6.25, 2.5, 1.25, 0.5, 0.25, and 0.05 nM l Substrate: 100 µM PREPARATION AND STORAGE 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 freeze­thaw cycles. l 6 months from date of receipt, ­20 to ­70 °C as supplied. l 3 months, ­20 to ­70 °C under sterile conditions after opening. Rev. 2/6/2018 Page 1 of 2 Recombinant Human Serpin D1/Heparin Cofactor II Catalog Number: 3198-PI BACKGROUND 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 C­terminal His­tagged recombinant Serpin D1 had enhanced heparin effect, which was maintained in a plasma­based thrombin inhibition assay (2). Congenital Serpin D1 deficiency is a potential risk factor for thrombosis (3). The full­length human cDNA was expressed and the purified protein corresponded to the secreted form with anti­thrombin 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. Rev. 2/6/2018 Page 2 of 2 .