Recombinant Human THOP1 Protein, CF Summary
Lys2-Cys689, with an N-terminal Met and 6-His tag
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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||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.
- 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
- Dilute rhTHOP1 to 0.5 ng/µL in Assay Buffer.
- Dilute Substrate to 20 µM in Assay Buffer.
- 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.
- Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively, in kinetic mode for 5 minutes.
- 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).
- rhTHOP1: 0.025 µg
- Substrate: 10 µM
Background: Thimet Oligopeptidase/THOP1
Thimet Oligopeptidase/THOP1, also known as endopeptidase EC 22.214.171.124 (EP24.15), is a zinc peptidase of the M3 family that also includes neurolysin/EC 126.96.36.199 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.
- 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.
- Ray, K. et al. (2004) J. Biol. Chem. 279:20480.
- Saric, T. et al. (2004) J. Biol. Chem. 279:46723.
- Shivakumar, B.R. et al. (2005) Cell Biochem. Funct. 23:195.
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