Recombinant Human Neurolysin Protein, CF Summary
Product Specifications
Ser38-Pro704, with an N-terminal Met and 6-His tag
Analysis
Product Datasheets
Carrier Free
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.
3814-ZN
| 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 Procedure
- Assay Buffer: 25 mM Tris, 150 mM NaCl, pH 7.5
- Recombinant Human Neurolysin (rhNeurolysin) (Catalog # 3814-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 rhNeurolysin to 5 µg/mL in Assay Buffer.
- Dilute Substrate to 50 µM in Assay Buffer.
- Load into a black well plate 50 µL of 5 µg/mL rhNeurolysin, and start the reaction by adding 50 µL of 50 µM Substrate. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of 50 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).
- rhNeurolysin: 0.25 µg
- Substrate: 25 µM
Reconstitution Calculator
Background: Neurolysin
Neurolysin, also known as Oligopeptidase M (mitochondrial peptidase), soluble angiotensin II-binding protein and endopeptidase EC 3.4.24.16 (1, 2), is a homologue of Thimet Ologopeptidase (THOP1), a zinc peptidase of the M3 family that also includes mitochondrial intermediate peptidase. Neurolysin expresses in two forms with the difference of an N-terminal transit peptide that targets the localization to mitochondria (1). The shorter form exists in cytosol. The recombinant human Neurolysin is expressed without the transit peptide and is isolated from the cytosol. Like THOP1, Neurolysin is capable of cleaving a number of vasoactive peptides such as bradykinin and neurotensin (3). All known substrates of THOP1 and Neurolysin contain 17 or fewer amino acids.
- Serizawa, A. et al. (1995) J. Biol. Chem. 270:2092.
- Barett, A.J. and J.M. Chen (2004) in Handbook of Proteolytic Enzymes (ed. Barrett, A.J. et al.) pp. 356, Elsevier Academic Press, San Diego.
- Norman, M.U. et al. (2003) Am. J. Physiol. Heart Circ. Physiol. 284:H1978.
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