Recombinant Human Serpin A3 Protein, CF

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Product Details

Summary
Reactivity HuSpecies Glossary
Applications Inhibition Activity
Format
Carrier-Free

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Recombinant Human Serpin A3 Protein, CF Summary

Details of Functionality
Measured by its ability to inhibit chymotrypsin cleavage of a fluorogenic peptide substrate MCA-RPKPVE-Nval-WRK(Dnp)-NH2 (Catalog # ES002). The IC50 value is <8 nM, as measured under the described conditions.
Source
Mouse myeloma cell line, NS0-derived human Serpin A3/alpha 1-Antichymotrypsin protein
Asn26-Ala423, with a C-terminal 10-His tag
Accession #
N-terminal Sequence
Asn26
Protein/Peptide Type
Recombinant Enzymes
Gene
SERPINA3
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

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
60-65 kDa, reducing conditions
Publications
Read Publications using
1295-PI in the following applications:

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 reconstitution.
Buffer
Lyophilized from a 0.2 μm filtered solution in HEPES and NaCl.
Purity
>90%, by SDS-PAGE under reducing conditions and visualized by silver stain
Reconstitution Instructions
Reconstitute at 100 μg/mL in sterile 25 mM HEPES and 150 mM NaCl, pH 7.8.
Assay Procedure
  • Assay Buffer: 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
  • Recombinant Human Serpin A3/ alpha 1-Antichymotrypsin (rhSerpin A3) (Catalog # 1295-PI)
  • Chymotrypsin (Sigma, Catalog # 3142), 40-80 units/mg, prepare a 1 mg/mL stock in 1 mM HCl
  • Substrate: MCA-Arg-Pro-Lys-Pro-Val-Glu-NVAL-Trp-Arg-Lys(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. Dilute Chymotrypsin to 0.6 µg/mL in Assay Buffer.
  2. Prepare a curve of rhSerpin A3 (MW: 46,373 kDa) in Assay Buffer. Make the following serial dilutions including the most concentrated dilution possible: (most concentrated), 1000, 500, 250, 175, 120, 80, 40, 20, and 5 nM.
  3. Mix equal volumes of rhSerpin A3 curve dilutions and 0.6 µg/mL Chymotrypsin in microtubes. Include two enzyme controls of equal volumes of Assay Buffer and 0.6 µg/mL Chymotrypsin.
  4. Incubate reaction mixtures at 37 °C for 30 minutes.
  5. Dilute incubated reaction mixtures by five fold in Assay Buffer.
  6. Dilute Substrate to 20 µM in Assay Buffer.
  7. In a plate load 50 µL of the diluted reaction mixtures to wells, 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 rhSerpin A3 by ploting RFU/min (or specific activity) vs concentration with 4-PL fitting.
  10. The specific activity for Chymotrypsin 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:
  • Chymotrypsin: 0.003 µg
  • rhSerpin A3: (most concentrated), 50, 25, 12.5, 8.75, 6, 4, 2, 1 and 0.25 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 Human Serpin A3 Protein, CF

  • AACTMGC88254
  • ACTGIG25
  • alpha 1-Antichymotrypsin
  • alpha-1-antichymotrypsin
  • antitrypsin), member 3
  • Cell growth-inhibiting gene 24/25 protein
  • growth-inhibiting protein 24
  • growth-inhibiting protein 25
  • member 3
  • serine (or cysteine) proteinase inhibitor, clade A (alpha-1 antiproteinase
  • serine (or cysteine) proteinase inhibitor, clade A, member 3
  • Serpin A3
  • serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin)

Background

Serpin A3 is a member of the Serpin superfamily of the serine protease inhibitors (1). It is synthesized primarily in the liver and secreted as one of the most abundant serpins in plasma (2). It is known to inhibit several serine proteases including chymotrypsin, cathepsin G, chymase, kallikrein 3/prostate specific antigen, and unidentified ectoenzymes that process pro-macrophage stimulating protein (1‑5). Serpin A3 is a major constituent of the plaques associated with Alzheimer’s disease and an inhibitor of amyloid beta peptide degradation (1‑6). Deficiency in Serpin A3 activity due to a point mutation (Leu55Pro) is associated with chronic obstructive pulmonary disease (7). Human Serpin A3 is synthesized as a 423 amino acid precursor (8, 9). The mature protein is secreted and has two forms that differ in their N‑termini (10). One of the forms starting at Asn26 was expressed and purified.

  1. Silverman, G.A. et al. (2001) J. Biol. Chem. 276:33293.
  2. Kalsheker, N. et al. (2002) Biochem. Soc. Trans. 30:93.
  3. Duranto, J. et al. (1998) Biochemistry 37:11239.
  4. Hsieh, M.-C. and B.S. Cooperman (2002) Biochemistry 41:2990.
  5. Skeel, A. and E.J. Leonard (2001) J. Biol. Chem. 276:21932.
  6. Abraham, C.R. et al. (2000) Ann. N. Y. Acad. Sci. 920:245.
  7. Gooptu, B. et al. (2000) Proc. Natl. Acad. Sci. USA 97:67.
  8. Chandra, T. et al. (1983) Biochemistry 22:5055.
  9. Morii, M. and J. Travis (1983) J. Biol. Chem. 258:12749.
  10. Lindmark, B. et al. (1989) Biochim. Biophys. Acta 997:90.

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Gene Symbol SERPINA3
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