Recombinant Human Serpin A3 Protein, CF Summary
Asn26-Ala423, with a C-terminal 10-His tag
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.
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.
|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.
- 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
- Dilute Chymotrypsin to 0.6 µg/mL in Assay Buffer.
- 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.
- 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.
- Incubate reaction mixtures at 37 °C for 30 minutes.
- Dilute incubated reaction mixtures by five fold in Assay Buffer.
- Dilute Substrate to 20 µM in Assay Buffer.
- 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.
- Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively, in kinetic mode for 5 minutes.
- Derive the 50% inhibiting concentration (IC50) for rhSerpin A3 by ploting RFU/min (or specific activity) vs concentration with 4-PL fitting.
- 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
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.
- Silverman, G.A. et al. (2001) J. Biol. Chem. 276:33293.
- Kalsheker, N. et al. (2002) Biochem. Soc. Trans. 30:93.
- Duranto, J. et al. (1998) Biochemistry 37:11239.
- Hsieh, M.-C. and B.S. Cooperman (2002) Biochemistry 41:2990.
- Skeel, A. and E.J. Leonard (2001) J. Biol. Chem. 276:21932.
- Abraham, C.R. et al. (2000) Ann. N. Y. Acad. Sci. 920:245.
- Gooptu, B. et al. (2000) Proc. Natl. Acad. Sci. USA 97:67.
- Chandra, T. et al. (1983) Biochemistry 22:5055.
- Morii, M. and J. Travis (1983) J. Biol. Chem. 258:12749.
- Lindmark, B. et al. (1989) Biochim. Biophys. Acta 997:90.
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.
Citations: Showing 1 - 3
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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.
Sample Types: Recombinant Protein
Enzymatic properties of human kallikrein-related peptidase 12 (KLK12).
Authors: Memari</LastName><ForeNam N</Initial, Memari N, Jiang W, Diamandis EP, Luo LY
Biol. Chem., 2007;388(4):427-35.
Sample Types: Recombinant Protein
Applications: Enzyme Assay
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.
Sample Types: Protein
Applications: Enzyme Assay
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