Recombinant Mouse Serpin A3N Protein, CF

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

Recombinant Mouse Serpin A3N Protein, CF Summary

• Details of Functionality: Measured by its ability to inhibit Granzyme B cleavage of tert-butoxycarbonyl-Ala-Ala-Asp-thiobenzyl ester (Boc-AAD-SBzl). The IC₅₀ is <25 nM, as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived mouse Serpin A3N protein
  Phe21-Lys418, with a C-terminal 6-His tag
• Accession #: Q91WP6
• N-terminal Sequence: Phe21
• Protein/Peptide Type: Recombinant Enzymes
• Gene: Serpina3n
• Purity: >85%, 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: 46 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: 59 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: >85%, by SDS-PAGE under reducing conditions and visualized by silver stain.
• Assay Procedure:
  • Assay Buffer: 50 mM Tris, pH 7.5
  • Activation Buffer: 50 mM MES, 50 mM NaCl, pH 5.5
  • Recombinant Mouse Serpin A3N (rmSerpin A3N) (Catalog # 4709-PI)
  • Recombinant Human Granzyme B (rhGranzyme B) (Catalog # 2906-SE)
  • Recombinant Mouse Active Cathepsin C/DPPI (rmCathepsin C) (Catalog # 2336-CY)
  • E-64 (Sigma, Catalog # E-3132), 1 mM stock in DMSO
  • DTNB (5,5’-dithio-bis (2-nitrobenzoic acid) (Sigma, Catalog # D-8130), 10 mM stock in DMSO
  • Substrate: tert-Butoxycarbonyl-Ala-Ala-Asp-thiobenzyl ester (SM Biochemicals LLC, Catalog # SMSB05), 10 mM stock in DMSO
  • 96-well Clear Plate (Costar, Catalog # 92592)
  • Plate reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Activate rhGranzyme B by diluting to 100 µg/mL with 10 µg/mL of rmCathepsin C in Activation Buffer.
  2. Incubate at 37 °C for 4 hours.
  3. Stop reaction with E-64 at final a concentration of 10 µM in Activation Buffer.
  4. Prepare a curve of rmSerpin A3N (MW: 45551 Da) in Assay Buffer. Make the following serial dilutions: 5000, 2500, 1000, 800, 650, 500, 250, 125, 50, and 10 nM.
  5. Dilute activated rhGranzyme B to 12.5 µg/mL in Assay Buffer.
  6. Combine 20 µL of 12.5 µg/mL rhGranzyme B with 20 µL of the rmSerpin A3N serial curve dilutions. Include two enzyme controls of 20 µL of 12.5 µg/mL rhGranzyme B with 20 µL Assay Buffer.
  7. Incubate mixtures at room temperature for 30 minutes.
  8. Dilute mixtures by adding 460 µL Assay Buffer to each.
  9. Dilute Substrate to 200 µM containing 200 µM of DTNB in Assay Buffer.
  10. In a plate load 50 µL of the diluted mixtures into wells.
  11. Start the reaction by adding 50 µL of 200 µM Substrate mixture.
  12. Read at a wavelength of 405 nm in kinetic mode for 5 minutes.
  13. Derive the 50% inhibiting concentration (IC₅₀) for rmSerpin A3N by plotting OD/min (or specific activity) vs. concentration with 4-PL fitting.
  14. The specific activity for rhGranzyme B at each point may be determined using the following formula (if needed):

  Specific Activity (pmol/min/µg) = (Adjusted Vmax* (OD/min) x well volume (L) x 10^12 pmol/M) / (ext. coeff** (M^-1cm^-1) x path corr.*** (cm) x amount of enzyme (µg))

  *Adjusted for Substrate Blank

  **Using the extinction coefficient 13260 M^-1cm^-1

  ***Using the path correction 0.32 cm

  Note: the output of many spectrophotometers is in mOD.

  Per Well:
  • rhGranzyme B: 0.025 µg
  • rmSerpin A3N curve: 100, 50, 20, 16, 13, 10, 5, 2.5, 1, 0.2, and 0 nM
  • Substrate: 100 µM
  • DTNB: 100 µM

Notes

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

Alternate Names for Recombinant Mouse Serpin A3N Protein, CF

• A1A;A1AT;AAT;Alpha-1-antitrypsin;alpha1AT;nNIF;PI;PI1;PRO2275;SERPINA1
• Serpin A3N
• Spi2

Background

Serpin A3N is a serine protease inhibitor that is structurally related to alpha 1-antichymotrypsin encoded by the SERPINA3 gene (1). Serpin A3N is highly expressed in brain, testis, lung, thymus, and spleen (2). Serpin A3N secreted by Sertoli cells may regulate the activity of locally produced Granzyme B (3). Granzyme B inhibition by Serpin A3N may therefore regulate Granzyme B-mediated killing by cytotoxic lymphocytes, providing a means to disable cell-mediated immune responses.

1. Forsyth, S. et al. (2003) Genomics 81:336.
2. Horvath, A. J. et al. (2004) J. Mol. Evol. 59:488.
3. Hirst, C. E. et al. (2001) Mol. Hum. Reprod. 7:1133.

Publications for Serpin A3N (4709-PI)(4)

Publications using 4709-PI

• H Kim, K Leng, J Park, AG Sorets, S Kim, A Shostak, RJ Embalabala, K Mlouk, KA Katdare, IVL Rose, SM Sturgeon, EH Neal, Y Ao, S Wang, MV Sofroniew, JM Brunger, DG McMahon, MS Schrag, M Kampmann, ES Lippmann Reactive astrocytes transduce inflammation in a blood-brain barrier model through a TNF-STAT3 signaling axis and secretion of alpha 1-antichymotrypsin Nature Communications, 2022-11-02;13(1):6581. 2022-11-02 [PMID: 36323693] (Bioassay, Mouse)
• YT Ho, T Shimbo, E Wijaya, T Kitayama, S Takaki, K Ikegami, K Miyashita, Y Ouchi, E Takaki, R Yamamoto, Y Kaneda, K Tamai Longitudinal single cell transcriptomics reveals a role for Serpina3n-mediated resolution of inflammation in a mouse colitis model Cellular and Molecular Gastroenterology and Hepatology, 2021-04-13;0(0):. 2021-04-13 [PMID: 33862275] (Bioassay, Mouse)
• HT Zhang, P Wang, Y Li, MM Zhang SerpinA3n affects ovalbumin (OVA)-induced asthma in neonatal mice via the regulation of collagen deposition and inflammatory response Respiratory physiology & neurobiology, 2021-02-17;288(0):103642. 2021-02-17 [PMID: 33609775] (Bioassay, Mouse)
• Vicuna L, Strochlic D, Latremoliere A, Bali K, Simonetti M, Husainie D, Prokosch S, Riva P, Griffin R, Njoo C, Gehrig S, Mall M, Arnold B, Devor M, Woolf C, Liberles S, Costigan M, Kuner R The serine protease inhibitor SerpinA3N attenuates neuropathic pain by inhibiting T cell-derived leukocyte elastase. Nat Med, 2015-04-27;21(5):518-23. 2015-04-27 [PMID: 25915831] (Bioassay, In Vivo, Mouse)

Bioinformatics

• Gene Symbol: Serpina3n
• Uniprot:
  • Q91WP6(Mouse)