Recombinant Mouse Serpin A1a Protein, CF

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

Recombinant Mouse Serpin A1a 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 is <5 nM, as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived mouse Serpin A1a protein
  Glu25-Lys413, with a C-terminal 6-His tag
• Accession #: AAH11040
• N-terminal Sequence: Glu25
• Protein/Peptide Type: Recombinant Enzymes
• Gene: Serpina1a
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Inhibition Activity
• Theoretical MW: 44 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: 50-60 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 Tris and NaCl.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
• Assay Procedure:
  • Assay Buffer: 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, 0.05% Brij-35 (w/v), pH 7.5 (TCNB)
  • Trypsin Buffer: 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, pH 7.5 (TCN)
  • Recombinant Mouse Serpin A1a (rmSerpin A1a) (Catalog # 7690-PI)
  • Trypsin, from bovine pancreas (Sigma, Catalog # T1426)
  • Substrate: MCA-RPKPVE-NVal-WRK(DNP)-NH₂ (Catalog # ES002), 2 mM stock in DMSO
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Prepare a curve of rmSerpin A1a (MW 44,000 Da). Make the following serial dilutions in Assay Buffer: 3600, 1800, 900, 450, 225, 112.5, 56.25, 28.13, and 14.1 nM.
  2. Dilute Trypsin to 5.0 µg/mL in Trypsin Buffer.
  3. Combine equal volumes of dilute rmSerpin A1a and Trypsin. Include a control containing equal volumes of Assay Buffer and Trypsin.
  4. Incubate reactions at room temperature for 30 minutes.
  5. Dilute incubated reaction mixtures 25-fold in Assay Buffer.
  6. Dilute Substrate to 20 µM in Assay Buffer.
  7. Load 50 µL of the incubated reactions into the wells of a black well plate, 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 of rmSerpin A1a (IC₅₀) 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: 0.005 µg (2 nM)
  • rmSerpin A1a: 36, 18, 9, 4.5, 2.25, 1.125, 0.563, 0.281, and 0.141 nM
  • ES002 : 10 µM

Notes

Coomassie is a registered trademark of Imperial Chemical Industries Ltd.

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

Alternate Names for Recombinant Mouse Serpin A1a Protein, CF

• A1A
• A1AT
• AAT
• Aat2
• alpha1AT
• Dom1
• nNIF
• PI
• PI1
• PRO2275
• Serpin A1a
• Spi1-1

Background

Serpin A1a, also known as alpha-1-antitrypsin, belongs to the serpin superfamily of proteinase inhibitors. It is one of five alpha 1-proteinase inhibitors produced in mice. Serpin A1a is produced in the liver, then secreted into the bloodstream. It inhibits a number of serine proteases, including elastase, trypsin, plasmin, and thrombin (1, 2). Serpin A1a’s major function is to protect the lungs from neutrophil elastase. Serpin A1a deficiency can cause complications such as emphysema, and chronic obstructive pulmonary disease (COPD).

1. Gettins, P.G. (2002) Chem. Rev. 102:4751.
2. Kalsheker N.A. (1989) Biosci. Rep. 9:129. 3. Ritchie, R.F. et al. (2000) J. Clin. Lab. Anal. 14:284.

Bioinformatics

• Gene Symbol: Serpina1a
• Uniprot:
  • AAH11040()