Recombinant Mouse TRH-degrading Ectoenzyme/TRHDE Protein, CF Summary
Arg64-His1025, with an N-terminal 6-His tag
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 Tris, 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 Buffer: 25 mM Tris, 200 mM NaCl, pH 7.0
- Recombinant Mouse TRH‑degrading Ectoenzyme/TRHDE (rmTRHDE) (Catalog # 2985-ZN)
- Recombinant Human DPPIV/CD26 (rhCD26) (Catalog # 9168-SE)
- Substrate: Pyr-His-Pro-AMC (Bachem, Catalog # I-1440), 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 rmTRHDE to 2 µg/mL in Assay Buffer.
- Dilute Substrate to 20 µM in Assay Buffer.
- Mix equal volumes of rmTRHDE and Substrate for final concentrations of 1 µg/mL and 10 µM respectively. Include a Substrate Blank containing Assay Buffer and Substrate.
- Incubate mixtures for 5 minutes at room temperature.
- Boil mixtures at 100 °C immediately after the incubation to stop the reaction.
- Spin down vials and gently vortex.
- Dilute rhCD26 to 1 µg/mL in Assay Buffer.
- In a plate load 50 µL of 1 µg/mL rhCD26 followed by adding 50 µL of the boiled samples to each well containing rhCD26.
- Incubate plate at room temperature for 10 minutes.
- Read at excitation and emission wavelengths of 380 nm and 460 nm, respectively, in endpoint mode.
- Calculate specific activity:
Specific Activity (pmol/min/µg) =
|Adjusted Fluorescence* (RFU) x Conversion Factor** (pmol/RFU)|
|Incubation time (min) x amount of enzyme (µg)|
*Adjusted for Substrate Blank
**Derived using calibration standard 7-amino, 4-Methyl Coumarin (Sigma, Catalog # A-9891).
- rmTRHDE: 0.05 µg
- rhCD26: 0.05 µg
- Substrate: 5 µM
Background: TRH-degrading Ectoenzyme/TRHDE
TRHDE, also known as pyroglutamyl peptidase II and thyroliberinase, is a metalloprotease that specifically removes pyroglutamate from thyrotropin-releasing hormone, a tripeptide of L-pyroglutamyl-L-histidyl-L-prolineamide. TRH functions as a hypothalamic hypophysiotropic neuropeptide and neurotransmitter/neuromodulator within the central nervous system (1). Inhibitors of TRHDE have potential applications as research and therapeutic agents because TRHDE inactivates TRH (2). TRHDE is a type II transmembrane protein and a soluble form is also present in the serum (1). The recombinant mouse TRHDE corresponds to the ectodomain of the enzyme. Its amino acid sequence is 97% and 95% identical to that of rat and human.
- Schmitmeier, S. et al. (2002) Eur. J. Biochem. 269:1278.
- Kelly, J.A. et al. (2005) Biochem. J. 389:569.
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