Recombinant E. coli Ecotin Protein, CF Summary
Product Specifications
Ala21-Arg162, with a C-terminal 6-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.
1328-PI
| Formulation | Lyophilized from a 0.2 μm filtered solution in Tris and NaCl. |
| Reconstitution | Reconstitute at 100 μg/mL in sterile 25 mM Tris and 350 mM NaCl, pH 8.5. |
| 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, 0.15 M NaCl, 10 mM CaCl2, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
- Recombinant E. coli Ecotin (reEcotin) (Catalog # 1328-PI)
- Trypsin (Sigma, Catalog # T-1426)
- Fluorogenic Peptide 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)
- Plate Reader (Model: SpectraMax Gemini by Molecular Devices) or equivalent
- Dilute Trypsin to 0.25 μg/mL in Assay Buffer.
- Prepare a curve of reEcotin (MW: 17,042 Da) in Assay Buffer. Make serial dilutions of: 200, 100, 40, 20, 16, 10, 4 and 1.6 nM.
- Combine equal volumes of the reEcotin curve dilutions and the diluted active Trypsin. Include a control (in duplicate) containing Assay Buffer and the diluted active Trypsin.
- Incubate mixtures at room temperature for 30 minutes.
- Perform 1:5 dilutions of reaction mixture with Assay Buffer.
- Dilute fluorogenic peptide substrate to 20 μM in Assay Buffer.
- Load in a black well 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% inhibition concentration (IC50) for reEcotin 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).
- Trypsin: 0.00125 μg
- reEcotin: 10, 5.0, 2.0, 1.0, 0.8, 0.5, 0.2, 0.08 nM
- Substrate: 10 μM
Reconstitution Calculator
Background: Ecotin
Ecotin is a potent and general inhibitor of serine proteases with diverse substrate specificities (1). It strongly inhibits trypsin, chymotrypsin, elastase, factor Xa, plasma kallikrein, u-plasminogen activator, granzyme B, and factor XIIa. Immobilized Ecotin has been used to affinity-purify recombinant trypsinogen, indicating that it may also be used to purify additional serine protease zymogens (2). Compared to other serine protease inhibitors such as members of the serpin family, the reactive site of Ecotin is Met104 (P1) (3). Ecotin is synthesized as a 162 amino acid precursor with a 20 amino acid signal peptide necessary to direct it to the periplasmic space. The mature protein (residues 21‑162) is expressed and purified.
Citation for Recombinant E. coli Ecotin 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.
1 Citation: Showing 1 - 1
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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., 2006-03-03;449(1):164-70.
Species: Human
Sample Types: Protein
Applications: Enzyme Assay
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