Recombinant Human Serpin A3 Protein, CF

Catalog # Availability Size / Price Qty
1295-PI-010
R&D Systems Recombinant Proteins and Enzymes
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Recombinant Human Serpin A3 Protein, CF Summary

Product Specifications

Purity
>90%, 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.
Activity
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
Analysis
Asn26
Predicted Molecular Mass
46 kDa
SDS-PAGE
60-65 kDa, reducing conditions

Product Datasheets

Carrier Free

What does CF mean?

CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.

What formulation is right for me?

In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.

1295-PI

Formulation Lyophilized from a 0.2 μm filtered solution in HEPES and NaCl.
Reconstitution Reconstitute at 100 μg/mL in sterile 25 mM HEPES and 150 mM NaCl, pH 7.8.
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 reconstitution.

Assay Procedure

Materials
  • 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

Reconstitution Calculator

Reconstitution Calculator

The reconstitution calculator allows you to quickly calculate the volume of a reagent to reconstitute your vial. Simply enter the mass of reagent and the target concentration and the calculator will determine the rest.

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Background: Serpin A3/alpha 1-Antichymotrypsin

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.

References
  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.
Entrez Gene IDs
12 (Human)
Alternate Names
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)

Citations for Recombinant Human Serpin A3 Protein, CF

R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.

3 Citations: Showing 1 - 3
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  1. Mass spectrometry-based determination of Kallikrein-related peptidase 7 (KLK7) cleavage preferences and subsite dependency
    Authors: LM Silva, T Stoll, T Kryza, CR Stephens, ML Hastie, HF Irving-Rod, Y Dong, JJ Gorman, JA Clements
    Sci Rep, 2017;7(1):6789.
    Species: N/A
    Sample Types: Recombinant Protein
    Applications: Bioassay
  2. Enzymatic properties of human kallikrein-related peptidase 12 (KLK12).
    Authors: Memari&lt;/LastName&gt;&lt;ForeNam N&lt;/Initial, Memari N, Jiang W, Diamandis EP, Luo LY
    Biol. Chem., 2007;388(4):427-35.
    Species: Human
    Sample Types: Recombinant Protein
    Applications: Enzyme Assay
  3. Mannan-binding lectin-associated serine protease 3 cleaves synthetic peptides and insulin-like growth factor-binding protein 5.
    Authors: Cortesio CL, Jiang W
    Arch. Biochem. Biophys., 2006;449(1):164-70.
    Species: N/A
    Sample Types: Protein
    Applications: Enzyme Assay

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Fluorogenic Peptide Substrates

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