Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
Dilute rhNeprilysin-2 to 1 µg/mL in Assay Buffer.
Dilute Substrate to 20 µM in Assay Buffer.
Load into a black well plate 50 µL of 1 µg/mL rhNeprilysin-2, and start the reaction by adding 50 µL of 20 µM Substrate. Include a Substrate Blank containing 50 µL of 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).
rhNeprilysin-2: 0.050 µg
Substrate: 10 µM
Neprilysin-2 is a zinc protease of the neprilysin (NEP) family, which also includes NEP, ECE-2, PEX, DINE, Kell and several NEP-like proteins (1). It is encoded by the MMEL1 gene in the human genome and is also known as soluble secreted endopeptidase (SEP), Neprilysin-like 1 and Neprilysin-2 (2). Highly expressed in testis, the cDNA predicted a type II transmembrane protein with a short cytoplasmic tail and a large ectodomain. Both membrane-bound and soluble forms were comparable with regard to model substrates, pH optima and inhibitor profiles. The ectodomain of human Neprilysin-2/MMEL1 was expressed with an N-terminal His tag and purified.
Turner, A.J. et al. (2001) BioEssays 23:261.
Carpentier, M. et al. (2004) in Handbook of Proteolytic Enzymes. Barrett, A.J. et al. eds. p. 426.
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.
Have you used Recombinant Human Neprilysin-2/MMEL1 Protein, CF?
Submit a review and receive a $25US/€18/£15/$25CAN amazon gift card if you include an image - $10US/€7/£6/$10CAN Amazon card for reviews without an image. Limited to verified customers in USA, Canada and Europe.