Recombinant Human Serpin A5 Protein, CF
Recombinant Human Serpin A5 Protein, CF Summary
Product Specifications
His20-Pro406, 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.
1266-PI
| Formulation | Lyophilized from a 0.2 μm filtered solution in MES and NaCl. |
| Reconstitution | Reconstitute at 100 μg/mL in sterile 25 mM MES, 150 mM NaCl, pH 6.5. |
| 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 Human Serpin A5/Protein C Inhibitor (rhSerpin A5) (Catalog # 1266-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)
- 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 A5 (MW: 45,003 Da) in Assay Buffer. Make the following serial dilutions: 200, 100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.781, and 0.391 nM.
- Mix equal volumes of rhSerpin A5 curve dilutions and Thrombin/Heparin mixture. Include a control (in duplicate) containing equal volumes of Assay Buffer and diluted Thrombin/Heparin mixture.
- Incubate reaction mixtures at room temperature for 30 minutes.
- After incubation, dilute the reaction mixtures by 1/5 in Assay Buffer.
- Dilute Substrate to 200 µM in Assay Buffer.
- In a plate load 50 µL of the diluted reaction mixtures to wells, 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 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: 0.002 µg
- rhSerpin A5: 10, 5, 2.5, 1.25, 0.625, 0.313, 0.156, 0.0781, 0.0391 and 0.0195 nM
- Substrate: 100 µM
Reconstitution Calculator
Background: Serpin A5/Protein C Inhibitor
Serpin A5 is the a member of the Serpin superfamily and inhibits a variety of serine proteases such as protein C, plasminogen activators, thrombin, factor Xa, several kallikreins and acrosin (1). Serpin A5 is synthesized in the liver and secreted in plasma. It is found in numerous steroid-responsive organs and has been detected in saliva, cerebral spinal fluid, amniotic fluid, tears and semen. Because of its protease targets and regulated expression patterns, Serpin A5 has been proposed to play a role in processes such as blood coagulation, fertilization and carcinogenesis (2, 3). Similar to Serpins C1 and D1, its thrombin inhibitory activity is enhanced by heparin.
- Silverman, G.A. et al. (2001) J. Biol. Chem. 276:33293.
- Palmier, D. et al. (2002) J. Biol. Chem. 277:40950.
- Geiger, M. et al. (1996) Immunopharmacology 32:53.
Citations for Recombinant Human Serpin A5 Protein, CF
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
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Citations: Showing 1 - 2
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Enzymatic properties of human kallikrein-related peptidase 12 (KLK12).
Authors: Memari</LastName><ForeNam N</Initial, Memari N, Jiang W, Diamandis EP, Luo LY
Biol. Chem., 2007-04-01;388(4):427-35.
Species: Human
Sample Types: Recombinant Protein
Applications: Enzyme Assay -
Mannan-binding lectin-associated serine protease 3 cleaves synthetic peptides and insulin-like growth factor-binding protein 5.
Authors: Cortesio CL, Jiang W
Arch. Biochem. Biophys., 2006-03-03;449(1):164-70.
Applications: Enzyme Assay
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