Recombinant Mouse Serpin A9/Centerin Protein, CF

• Reactivity: Mu
• Applications: Inhibition Activity
• Format: Carrier-Free

Recombinant Mouse Serpin A9/Centerin Protein, CF Summary

• Details of Functionality: Measured by its ability to inhibit trypsin cleavage of a fluorogenic peptide substrate, Mca-RPKPVE-Nval-WRK(Dnp)-NH₂ (Catalog # ES002). The IC₅₀ value is <7 nM, measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived mouse Serpin A9/Centerin protein
  Asn26-Met418, with an N-terminal 6-His tag
  Accession # Q9D7D2
• Accession #: Q9D7D2
• N-terminal Sequence: His
• Structure / Form: Monomer
• Protein/Peptide Type: Recombinant Enzymes
• Gene: Serpina9
• Purity: >90%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Inhibition Activity
• Theoretical MW: 45 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 40-50 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -70 °C as supplied.
  • 3 months, -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris and NaCl.
• Purity: >90%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Assay Procedure:
  • Assay Buffer: 50 mM Tris, 10 mM CaCl₂, 0.15 M NaCl, 0.05% (v/v) Brij-35, pH 7.5 (TCNB)
  • Recombinant Mouse Serpin A9/Centerin (rmSerpin A9) (Catalog # 5679-PI)
  • Recombinant Human Active Trypsin 3/PRSS3 (rhTrypsin 3) (Catalog # 3714-SE)
  • Fluorogenic Peptide Substrate: MCA-Arg-Pro-Lys-Pro-Val-Glu-NVAL-Trp-Arg-Lys(DNP)-NH₂ (Catalog # ES002)
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Dilute rhTrypsin 3 to 1 ng/µL in Assay Buffer.
  2. Prepare a curve of rmSerpin A9 (MW: 44,977 Da) in Assay Buffer. Make the following serial dilutions: 1200 nM, 600 nM, 300 nM, 150 nM, 75 nM, 37.5 nM, 18.8 nM, 9.4 nM, 4.7 nM, and 2.3 nM.
  3. Combine equal volumes of dilute rhTrypsin 3 and rmSerpin A9 at each concentration of the curve. Include two controls containing equal volumes of Assay Buffer and diluted rhTrypsin 3 without any rmSerpin A9.
  4. Incubate mixtures at 37 °C for 30 minutes.
  5. Dilute the reaction mixtures 5-fold with Assay Buffer.
  6. Dilute Substrate to 20 µM with Assay Buffer.
  7. Load in a plate 50 µL of the incubated mixtures, and start the reaction by adding 50 µL of 20 mM Substrate.
  8. Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively, in kinetic mode for 5 minutes.
  9. Derive the 50% inhibition concentration (IC₅₀) for rmSerpin A9 by plotting RFU/min (or specific activity) vs concentration with 4-PL fitting.
  10. Calculate specific activity of Trypsin at each point using the following formula (if needed):

  Specific Activity (pmoles/min/µg) = (Adjusted Vmax* (RFU/min) x Conversion Factor** (pmole/RFU)) / (amount of enzyme (µg))

  *Adjusted for Substrate Blank

  **Derived using calibration standard MCA-Pro-Leu-OH (Bachem, Catalog # M-1975).

  Per Well:
  • rhTrypsin 3: 0.005 µg (2.1 nM)
  • rmSerpin A9 curve: 60 nM, 30 nM, 15 nM, 7.5 nM, 3.8 nM, 1.9 nM, 0.94 nM, 0.47 nM, 0.23 nM, and 0.12 nM
  • Substrate: 10 µM

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Mouse Serpin A9/Centerin Protein, CF

• Centerin
• GCET1
• Serpin A9
• SERPINA11
• SERPINA11b

Background

Serpin A9, also known as Centerin, is a member of the Serpin superfamily of serine protease inhibitors (1). Serpins are the most broadly distributed superfamily of protease inhibitors. They are known to inhibit serine proteases and some are known to inhibit caspases and papain-like cysteine proteases (1). Recombinant mouse Serpin A9 shows inhibition towards trypsin, thrombin, and plasmin and binds DNA and heparin (2). Its expression is limited to germinal center B cells and lymphoid malignancies with germinal center B-cell maturation (3). New findings suggest that Serpin A9 could be used as a potential diagnostic marker in the recognition of various germinal center derived lymphomas (4).

1. Law, R., et al. (2006) Genome Biol. 7:216.
2. Paterson, M.A., et al. (2007) Biochem J. 405:489.
3. Pan, Z., et al. (2003) Am. J. Pathol. 163:135.
4. Montes-Moreno, S. et al. (2008) Blood. 111:351.
   
   

Bioinformatics

• Gene Symbol: Serpina9
• Uniprot:
  • Q9D7D2()