Recombinant Mouse Serpin A3N Protein, CF

R&D Systems | Catalog # 4709-PI

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Key Product Details

  • R&D Systems NS0-derived Recombinant Mouse Serpin A3N Protein (4709-PI)
  • Quality control testing to verify active proteins with lot specific assays by in-house scientists
  • All R&D Systems proteins are covered with a 100% guarantee

Source

NS0

Accession Number

Q91WP6

Applications

Inhibition Activity
View all Serpin A3N Proteins and Enzymes »

Product Specifications

Source

Mouse myeloma cell line, NS0-derived mouse Serpin A3N protein
Phe21-Lys418, with a C-terminal 6-His tag

Purity

>85%, by SDS-PAGE under reducing conditions and visualized by silver stain.

Endotoxin Level

<1.0 EU per 1 μg of the protein by the LAL method.

N-terminal Sequence Analysis

Phe21

Predicted Molecular Mass

46 kDa

SDS-PAGE

59 kDa, reducing conditions

Activity

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.

Formulation, Preparation, and Storage

4709-PI
Formulation Supplied as a 0.2 μm filtered solution in Tris and NaCl.
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.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.

Background: Serpin A3N

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.

References

  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.

Alternate Names

Spi2

Entrez Gene IDs

20716 (Mouse); 24795 (Rat)

Gene Symbol

SERPINA3N

UniProt

Q91WP6 (Mouse)

Additional Serpin A3N Products

Product Documents for Recombinant Mouse Serpin A3N Protein, CF

Certificate of Analysis

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Product Specific Notices for Recombinant Mouse Serpin A3N Protein, CF

For research use only

Citations for Recombinant Mouse Serpin A3N Protein, CF

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Protocols

View specific protocols for Recombinant Mouse Serpin A3N Protein, CF (4709-PI):

Materials
  • 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 1012 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

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