The peptide substrate contains a highly fluorescent 7-methoxycoumarin group that is efficiently quenched by resonance energy transfer to the 2,4-dinitrophenyl group. It can be used to measure the activities of peptidases that are capable of cleaving an amide bond between the fluorescent group and the quencher group, causing an increase in fluorescence. It is an excellent substrate for endothelin-converting enzyme-1 (ECE-1) and neprilysin. The cleavage site by ECE-1 is the peptide bond between Ala and Phe (Johnson, G.D. and Ahn, K., 2000, Anal. Biochem. 286:112). The substrate is derived from bradykinin. It is also an excellent substrate for ACE and active Cathepsin A and X/Z/P.
Formulation, Preparation and Storage
What does CF mean?
CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our
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
Stock solution at 4 mM or 6.26 mg/mL (0.16 mL) dimethyl sulfoxide (DMSO).
The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.
Stability & Storage:
Samples are stable for up to twelve months from date of receipt at -20° C to -70° C. The substrate can be aliquoted and stored -20° C to -70° C in a manual defrost freezer for six months. Avoid repeated freeze-thaw cycles. Protect from exposure to direct light
Plan Your Experiments
Use our spectra viewer to interactively plan your experiments,
assessing multiplexing options. View the excitation and emission spectra
for our fluorescent dye range and other commonly used dyes.
ACE activity was determined following incubation with intramolecularly quenched synthetic ACE specific substrate Mca-RPGFSAFK (Dnp)-OH (R&D systems). In the case of cell lysates, 10 µg of total protein was assayed for activity in a buffer with the following composition: 50 mM MES (4-morpholineethanesulphonic acid), 300 mM NaCl, 10 µm ZnCl2 and 0.01% Triton X-100, pH 6.5. Reaction was initiated by the addition of 5×10−5 M substrate. Where applicable, recombinant enzymes were used at a concentration of 0.01 µg per reaction. The fluorescence measurements were performed in the black microtiter plates (Costar) in a total volume of 100 µl. The plates were read using a fluorescence plate reader SpectraMax M5 (Molecular Devices) at an excitation wavelength 320 nm and emission wavelength 405 nm Fluorescence resulting from the substrate hydrolysis increased with time, and achieved maximum by one h with recombinant enzyme however with cell/tissue lysates the maximum fluorescence was observed by four hours of incubation. Therefore fluorescence recorded at one and four hours of reaction time was taken for calculation of percent enzyme inhibition, when using recombinant enzymes and cell/tissue lysates, respectively. ACE activity was defined as the ACE inhibitor, captopril-sensitive fluorescence, and were expressed as percent inhibition.
FAQs for Mca-RPPGFSAFK(Dnp)-OH Fluorogenic Peptide Substrate
Where does Cathepsin A cleave Mca-RPPGFSAFK(Dnp)-OH Fluorogenic Peptide Substrate, Catalog # ES005?
Although the QC assay conditions provided on our recombinant Cathepsin A datasheets should favor carboxypeptidase activity, it is possible that there is more than one site in the ES005 peptide recognized and cleaved by Cathepsin A. We have not performed verification experiments to confirm the cleavage site preferred in our reaction conditions.
Product Documents for Mca-RPPGFSAFK(Dnp)-OH Fluorogenic Peptide Substrate
Which Brands are Currently Available on bio‑techne.com? R&D Systems, Tocris Bioscience and ProteinSimple branded products are available to purchase through bio‑techne.com. ProteinSimple branded instruments are available to quote. ACD branded products will be available on bio‑techne.com in the near future. Novus Biologicals branded products are not currently available on bio‑techne.com and can be found at novusbio.com.
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