Recombinant E. coli Ecotin Protein, CF

R&D Systems | Catalog # 1328-PI

R&D Systems
100% Guarantee

Key Product Details

  • R&D Systems E. coli-derived Recombinant E. coli Ecotin Protein (1328-PI)
  • Quality control testing to verify active proteins with lot specific assays by in-house scientists
  • All R&D Systems proteins are covered with a 100% guarantee

Source

E. coli

Accession Number

NP_416713

Applications

Inhibition Activity
View all Ecotin Proteins and Enzymes »

Product Specifications

Source

E. coli-derived e. coli Ecotin protein
Ala21-Arg162, with a C-terminal 6-His tag

Purity

>95%, 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.

N-terminal Sequence Analysis

Ala21

Predicted Molecular Mass

17 kDa

SDS-PAGE

18 kDa, reducing conditions

Activity

Measured by its ability to inhibit trypsin cleavage of a fluorogenic peptide substrate, Mca-RPKPVE-Nval-WRK(Dnp)-NH2 (Catalog # ES002).
The IC50 value is <1.0 nM, as measured under the described conditions.

Formulation, Preparation, and Storage

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.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after reconstitution.

Calculators

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.

=
÷

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.

References

  1. McGrath, M.E. et al. (1995) Protein Sci. 4:141.
  2. Lengyel, Z. et al. (1998) Protein Expre. Purif. 12:291.
  3. McGrath, M.E. et al. (1991) J. Biol. Chem. 28:6620.

Gene Symbol

eco

UniProt

NP_416713

Additional Ecotin Products

Product Documents for Recombinant E. coli Ecotin Protein, CF

Certificate of Analysis

To download a Certificate of Analysis, please enter a lot or batch number in the search box below.

Note: Certificate of Analysis not available for kit components.

Download SDS

Product Specific Notices for Recombinant E. coli Ecotin Protein, CF

For research use only

Related Research Areas

Serine Proteases and Regulators

Citations for Recombinant E. coli Ecotin Protein, CF

Customer Reviews for Recombinant E. coli Ecotin Protein, CF

There are currently no reviews for this product. Be the first to review Recombinant E. coli Ecotin Protein, CF and earn rewards!

Have you used Recombinant E. coli Ecotin Protein, CF?

Submit a review and receive an Amazon gift card!

$25/€18/£15/$25CAN/¥2500 Yen for a review with an image

$10/€7/£6/$10CAN/¥1110 Yen for a review without an image

Submit a review
Amazon Gift Card

Protocols

View specific protocols for Recombinant E. coli Ecotin Protein, CF (1328-PI):

Materials
  • 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

  1. Dilute Trypsin to 0.25 μg/mL in Assay Buffer.
  2. 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.
  3. 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.
  4. Incubate mixtures at room temperature for 30 minutes.
  5. Perform 1:5 dilutions of reaction mixture with Assay Buffer.
  6. Dilute fluorogenic peptide substrate to 20 μM in Assay Buffer.
  7. Load in a black well plate 50 μL of the incubated mixtures, 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% inhibition concentration (IC50) for reEcotin by plotting RFU/min (or specific activity) vs. concentration with 4-PL fitting.
  10. 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
  • reEcotin: 10, 5.0, 2.0, 1.0, 0.8, 0.5, 0.2, 0.08 nM
  • Substrate: 10 μM

FAQs

No product specific FAQs exist for this product.

View all FAQs for Proteins and Enzymes