Recombinant Mouse Serpin A1c/alpha-1-Antitrypsin Protein, CF
Recombinant Mouse Serpin A1c/alpha-1-Antitrypsin Protein, CF Summary
Product Specifications
Glu25-Lys413, with a C-terminal 10-His tag
Analysis
Product Datasheets
Carrier Free
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.
2979-PI
| Formulation | Supplied as a 0.2 μm filtered solution in Sodium Acetate, NaCl and Glycerol. |
| 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 Procedure
- Assay Buffer: 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
- Recombinant Mouse Serpin A1c/ alpha 1‑Antitrypsin (rmSerpin A1c) (Catalog # 2979-PI)
- Trypsin (Sigma, Catalog # T-1426)
- Substrate: Mca-Arg-Pro-Lys-Pro-Val-Glu-NVAL-Trp-Arg-Lys(Dnp)-NH2 (Catalog # ES002), 2 mM in DMSO
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Dilute Trypsin to 0.25 ng/µL in Assay Buffer.
- Prepare a curve of rmSerpin A1c (MW: 44,709 Da) in Assay Buffer. Make the following serial dilutions: 2000, 1000, 500, 250, 125, 62.5, 31.25, 7.81 and 1.95 nM.
- Combine equal volumes of dilute Trypsin and rmSerpin A1c at each concentration of the curve. Include two controls containing equal volumes of Assay Buffer and diluted Trypsin.
- Incubate mixtures at room temperature for 30 minutes.
- Dilute the reaction mixtures five fold with Assay Buffer.
- Dilute Substrate to 20 µM with Assay Buffer.
- Load into a plate 50 µL of the incubated mixtures, 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 rmSerpin A1c by plotting RFU/min (or specific activity) vs. concentration with 4-PL fitting.
- The specific activity for Trypsin 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:- Trypsin: 0.00125 µg
- rmSerpin A1c curve: 100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.39 and 0.098 nM
- Substrate: 10 µM
Reconstitution Calculator
Background: Serpin A1c/alpha 1-Antitrypsin
Serpin A1 is the archetypal member of the Serpin superfamily of the serine protease inhibitors (1). As one of the most abundant proteinase inhibitors in the circulation, it is synthesized in the liver and secreted into the bloodstream with the major function to protect tissues against neutrophil elastase. Severe Serpin A1 deficiency leads to several clinical complications such as pulmonary emphysema, juvenile hepatitis, cirrhosis, and hepatocellular carcinoma (2). In contrast to a single Serpin A1 gene in the human genome, there is a cluster of five Serpin A1 genes in the mouse genome with high overall sequence identity but markedly divergent residues in the reactive center loop (3, 4). The purified rmSerpin A1c inhibits trypsin and chymotrypsin, but does not inhibit neutrophil elastase.
- Silverman, G.A. et al. (2001) J. Biol. Chem. 276:33293.
- Barbour, K.W. et al. (2002) Genomics 80:515.
- Forsyth, S. et al. (2003) Genomics 81:336.
- Puente, X.S. and Lopez-Otin, C. (2004) Genome Res. 14:609.
FAQs
-
Serpin A1 is a serine protease inhibitor. Does Mouse Serpin A1c inhibit all serine proteases?
Mouse Serpin A1c inhibits trypsin and chymotrypsin, but it does not inhibit neutrophil elastase.
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