Recombinant Mouse Serpin A1c/alpha-1-Antitrypsin Protein, CF

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

Recombinant Mouse Serpin A1c/alpha-1-Antitrypsin 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 <10 nM, as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived mouse Serpin A1c/alpha 1-Antitrypsin protein
  Glu25-Lys413, with a C-terminal 10-His tag
• Accession #: NP_033271
• N-terminal Sequence: Glu25
• Protein/Peptide Type: Recombinant Enzymes
• Gene: Serpina1c
• Purity: >95%, 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: 60 kDa, reducing conditions

Packaging, Storage & Formulations

• 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.
• Buffer: Supplied as a 0.2 μm filtered solution in Sodium Acetate, NaCl and Glycerol.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• 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 A1c/ alpha 1‑Antitrypsin (rmSerpin A1c) (Catalog # 2979-PI)
  • Trypsin (Sigma, Catalog # T-1426)
  • Substrate: Mca-Arg-Pro-Lys-Pro-Val-Glu-NVAL-Trp-Arg-Lys(Dnp)-NH₂ (Catalog # ES002) , 2 mM in DMSO
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Dilute Trypsin to 0.25 ng/µL in Assay Buffer.
  2. Prepare a curve of rmSerpin A1c (MW: 44,709 Da) in Assay Buffer. Make the following serial dilutions: 2000, 1000, 500, 250, 125, 62.5, 31.25, 7.81 and 1.95 nM.
  3. Combine equal volumes of dilute Trypsin and rmSerpin A1c at each concentration of the curve. Include two controls containing equal volumes of Assay Buffer and diluted Trypsin.
  4. Incubate mixtures at room temperature for 30 minutes.
  5. Dilute the reaction mixtures five fold with Assay Buffer.
  6. Dilute Substrate to 20 µM with Assay Buffer.
  7. Load into a plate 50 µL of the incubated mixtures, and start the reaction by adding 50 µL of 20 µM 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% inhibiting concentration (IC₅₀) for rmSerpin A1c by plotting RFU/min (or specific activity) vs. concentration with 4-PL fitting.
  10. The specific activity for Trypsin 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 MCA-Pro-Leu-OH (Bachem, Catalog # M-1975).

  Per Well:
  • Trypsin: 0.00125 µg
  • rmSerpin A1c curve: 100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.39 and 0.098 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 A1c/alpha-1-Antitrypsin Protein, CF

• A1A;A1AT;AAT;Alpha-1-antitrypsin;alpha1AT;nNIF;PI;PI1;PRO2275;SERPINA1
• alpha 1Antitrypsin
• Serpin A1c
• Spi1-3
• Spi1-6
• Wap3

Background

Serpin A1 is the archetypal member of the Serpin superfamily of the serine protease inhibitors (1). As one of the most abundant proteinase inhibitors in the circulation, it is synthesized in the liver and secreted into the bloodstream with the major function to protect tissues against neutrophil elastase. Severe Serpin A1 deficiency leads to several clinical complications such as pulmonary emphysema, juvenile hepatitis, cirrhosis, and hepatocellular carcinoma (2). In contrast to a single Serpin A1 gene in the human genome, there is a cluster of five Serpin A1 genes in the mouse genome with high overall sequence identity but markedly divergent residues in the reactive center loop (3, 4). The purified rmSerpin A1c inhibits trypsin and chymotrypsin, but does not inhibit neutrophil elastase.

1. Silverman, G.A. et al. (2001) J. Biol. Chem. 276:33293.
2. Barbour, K.W. et al. (2002) Genomics 80:515.
3. Forsyth, S. et al. (2003) Genomics 81:336.
4. Puente, X.S. and Lopez-Otin, C. (2004) Genome Res. 14:609.
   

Bioinformatics

• Gene Symbol: Serpina1c
• Uniprot:
  • NP_033271()