Recombinant Human THOP1 Protein, CF

Catalog # Availability Size / Price Qty
3439-ZN-010
R&D Systems Recombinant Proteins and Enzymes
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Recombinant Human THOP1 Protein, CF Summary

Product Specifications

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.
Activity
Measured by its ability to cleave a fluorogenic peptide substrate, (7-methoxycoumarin-4-yl)acetyl-Pro-Leu-Gly-Pro-D-Lys(2,4-dinitrophenyl)-OH or Mca-PLGPK(Dnp)-OH. The specific activity is >250 pmol/min/µg, as measured under the described conditions.
Source
E. coli-derived human Thimet Oligopeptidase/THOP1 protein
Lys2-Cys689, with an N-terminal Met and 6-His tag
Accession #
N-terminal Sequence
Analysis
Met
Predicted Molecular Mass
80 kDa
SDS-PAGE
80 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.

3439-ZN

Formulation Supplied as a 0.2 μm filtered solution in MES, NaCl and Glycerol.
Shipping The product is shipped with dry ice or equivalent. 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 opening.

Assay Procedure

Materials
  • Assay Buffer: 25 mM Tris, 150 mM NaCl, pH 7.5
  • Recombinant Human Thimet Oligopeptidase/THOP1 (rhTHOP1) (Catalog # 3439-ZN)
  • Substrate: MCA-Pro-Leu-Gly-Pro-D-Lys-(DNP)-OH (Bachem, Catalog # M-2270); 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 rhTHOP1 to 0.5 ng/µL in Assay Buffer.
  2. Dilute Substrate to 20 µM in Assay Buffer.
  3. Load 50 µL of the 0.5 ng/µL rhTHOP1 in a plate, and start the reaction by adding 50 µL of 20 µM Substrate. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of 20 µM Substrate.
  4. Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively, in kinetic mode for 5 minutes.
  5. Calculate specific activity:

     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:
  • rhTHOP1: 0.025 µg
  • 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: Thimet Oligopeptidase/THOP1

Thimet Oligopeptidase/THOP1, also known as endopeptidase EC 3.4.24.15 (EP24.15), is a zinc peptidase of the M3 family that also includes neurolysin/EC 3.4.24.16 and mitochondrial intermediate peptidase (1). Widely expressed by mammalian tissues and reported to present in different subcellular locations, THOP1 is primarily a cytoplasmic enzyme. It is capable of hydrolyzing a number of bioactive peptides and peptides released by the proteosome, limiting antigenic presentation by MHC class I molecules (1-3). THOP1 also interacts with angiotensin II type I receptor and bradykinin B2 receptor (4). The optimal activity of the purified THOP1 may or may not require the presence of a reducing agent, depending upon the source of the enzyme and the purification method used.

References
  1. Barrett, A.J. and J.-M. Chen (2004) in Handbook of Proteolytic Enzymes. Barrett, A.J. et al. eds. p. 352, Elsevier Academic Press, San Diego.
  2. Ray, K. et al. (2004) J. Biol. Chem. 279:20480.
  3. Saric, T. et al. (2004) J. Biol. Chem. 279:46723.
  4. Shivakumar, B.R. et al. (2005) Cell Biochem. Funct. 23:195.
Entrez Gene IDs
7064 (Human); 50492 (Mouse); 64517 (Rat)
Alternate Names
EC 3.4.24; EC 3.4.24.15; Endopeptidase 24.15; EP24.15; MEPD_HUMAN; MP78; thimet oligopeptidase 1; thimet oligopeptidase; THOP1; TOP

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