Recombinant Mouse Serpin D1/Heparin Cofactor II Protein, CF
Recombinant Mouse Serpin D1/Heparin Cofactor II Protein, CF Summary
Product Specifications
Glu24-Ser478, with a C-terminal 10-His tag
Analysis
Product Datasheets
Carrier Free
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.
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.
|
Assay Procedure
- 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).
- 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.
- 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.
FAQs
No product specific FAQs exist for this product, however you may
View all Proteins and Enzyme FAQsReviews for Recombinant Mouse Serpin D1/Heparin Cofactor II Protein, CF
There are currently no reviews for this product. Be the first to review Recombinant Mouse Serpin D1/Heparin Cofactor II Protein, CF and earn rewards!
Have you used Recombinant Mouse Serpin D1/Heparin Cofactor II Protein, CF?
Submit a review and receive an Amazon gift card.
$25/€18/£15/$25CAN/¥75 Yuan/¥2500 Yen for a review with an image
$10/€7/£6/$10 CAD/¥70 Yuan/¥1110 Yen for a review without an image
