Mca rppgfsafk dnp oh

Manufactured by R&D Systems

Mca-RPPGFSAFK(Dnp)-OH is a peptide compound that is commonly used in research applications. It serves as a substrate for enzymatic activity evaluation.

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13c6 15n2 lysine, by Merck Group (1 mentions) Flexmap 3d instrument, by Merck Group (1 mentions) Ala amc, by Bachem (1 mentions) Leu amc, by Bachem (1 mentions) Kta pure fplc, by GE Healthcare (1 mentions) Insulin, by Merck Group (1 mentions) Recombinant proteins of sars cov rbd 40150 v08b2, by Sino Biological (1 mentions) Sars cov 2 rbd proteins 40592 v08h, by Sino Biological (1 mentions) Drvyihpf, by GenScript (1 mentions) Synergy h1, by Synergy Software (1 mentions) Mca pro leu oh, by Bachem (1 mentions) Mca yvadapk dnp oh es007, by R&D Systems (1 mentions)

Close spelling variants of this product

MCA-RPPGFSAFK(DNP)-OH fluorogenic peptide substrate

6 protocols using mca rppgfsafk dnp oh

In Vitro Selection of IDE-Targeting Macrocyclic Inhibitors

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The in vitro selection of the DNA-templated library^{8 (link)} used 20 µg His₆-tagged mouse IDE immobilized on cobalt magnetic beads (Invitrogen). IDE inhibition was assayed using the fluorogenic peptide Mca-RPPGFSAFK(Dnp)-OH (R&D), confirmed using an anti-insulin antibody time-resolved FRET assay (Cysbio), and a LCMS assay for CGRP cleavage fragments in plasma^{9 (link)}. Macrocyclic inhibitors were synthesized by Fmoc-based solid-phase synthesis and purified by HPLC. LCMS quantitation of 6bK in biological samples was performed using 6bK synthesized with ¹³C₆,¹⁵N₂ lysine (Sigma-Aldrich).
Wild-type lean and DIO C57BL/6J age-matched male mice (Jackson Laboratories) were used at 14–16, and 24–26 weeks respectively (> 20 weeks of high-fat diet). Gcgr^−/− and Ide^−/− mice were fully backcrossed to the C57BL/6J line, bred from heterozygous mice, and used between 11 and 21 weeks. Animals were fasted overnight 14 h for all experiments, except for the insulin tolerance test, which required 5 h of fasting during the morning. Blood glucose was measured from tail nicks using AccuCheck (Aviva) meters. Trunk blood was obtained for plasma hormone measurements using the Multiplexed Mouse Metabolic Hormone panel (Milliplex, EMD Millipore) on a Luminex FlexMap 3D instrument.

Maianti J.P., McFedries A., Foda Z.H., Kleiner R.E., Du X.Q., Leissring M.A., Tang W.J., Charron M.J., Seeliger M.A., Saghatelian A, & Liu D.R. (2014). Anti-diabetic activity of insulin-degrading enzyme inhibitors mediated by multiple hormones. Nature, 511(7507), 94-98.

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Kinetic Analysis of NEP Activity

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The 20 μ assay was performed on low-volume black Costar 384-well plates at 25 C°. A working solution of 160 μM peptide substrate MCA-RPPGFSAFK(Dnp)-OH (R&D Systems) was prepared in 50 mM Tris-HCl pH7.8, 25 mM NaCl and 5 mM ZnCl2. 10 μl of NEP (R&D Systems) or NEP fusion polypeptide, diluted to 1, 2 or 4 nM in assay buffer, were transferred to plate. For determination of apparent K_M values various concentrations of substrate (0.078–80 nM in 2-fold dilutions) were added and the enzyme reaction started. The fluorescence increase was monitored with excitation at 320 nm and emission at 405 nm on an Envision Reader. Hydrolysis rates and apparent K_M values were calculated using XLFit^® software (IDBS).

Campos C.R., Kemble A.M., Niewoehner J., Freskgård P.O, & Urich E. (2020). Brain Shuttle Neprilysin reduces central Amyloid-β levels. PLoS ONE, 15(3), e0229850.

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Enzymatic Inhibition Assay Protocol

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Purified recombinant human enzymes were purchased from R&D Systems (Minneapolis, MN). The quenched peptide substrate for aminopeptidase N was Ala-AMC (Bachem 1410, Torrance, CA). The substrate for puromycin-sensitive aminopeptidase was Leu-AMC (Bachem 1240). The substrate for all remaining enzymes was MCA-RPPGFSAFK-Dnp-OH (R&D Systems). Each substrate was diluted to 100 μM in the assay buffer recommended for each enzyme by the supplier. To a black microtiter plate (Corning 3686), 5 μl of compound dilutions in assay buffer were aliquoted in triplicate. To these, 5 μl of diluted enzyme was added, followed by 10 μl of the quenched peptide substrate. The plates were incubated at 37°C until at least 10% substrate turnover or 5 μM product had formed. Fluorescence was measured using a Tecan Safire II (Excitation 320 nm/Emission 420 nm). Each plate included a standard curve of reaction product and positive (no enzyme) and negative (dimethyl sulfoxide vehicle) controls. Dose-response data were plotted and inhibition values (IC₅₀) determined by linear regression analysis using GraphPad Prism (GraphPad Software, La Jolla, CA).

Griggs D.W., Prinsen M.J., Oliva J., Campbell M.A., Arnett S.D., Tajfirouz D., Ruminski P.G., Yu Y., Bond B.R., Ji Y., Neckermann G., Choy R.K., de Hostos E, & Meyers M.J. (2016). Pharmacologic Comparison of Clinical Neutral Endopeptidase Inhibitors in a Rat Model of Acute Secretory Diarrhea. The Journal of Pharmacology and Experimental Therapeutics, 357(2), 423-431.

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Purification and Characterization of Recombinant Human IDE

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The expression and purification of glutathione-S-transferase tagged human IDE (GST-IDE in pGEX-6p-1 vector, kindly provided by Dr. Malcolm Leissring of the University of California at Irvine) were accomplished following a protocol^{26 (link)} provided by Dr. Leissring with some modifications. Expression of GST-IDE in Escherichia coli BL21 (DE3) CodonPlus RIL was induced with 50 μM IPTG overnight at 25 °C. The non-metalloprotease inhibitor phenylmethylsulfonyl fluoride was added prior to cracking the cells. GST-IDE was purified using a 5 mL GST Trap Fast Flow column on an ÄKTA Pure FPLC (GE Healthcare Life Sciences) and eluted with PBS containing 10 mM glutathione. After cleavage of the GST tag using GST PreScission protease, further purification of IDE was accomplished using a HiLoad 16/600 Superdex S-200 pg gel filtration column preequilibrated with PBS on the ÄKTA Pure FPLC. Purified protein concentration was determined by UV–vis spectroscopy using the molar extinction coefficient of IDE at 280 nm, ε_280nm = 113570 M⁻¹ cm⁻¹.^{42 (link)} Flash-frozen IDE aliquots containing 1% glycerol (v/v) were stored at −80 °C in PBS (pH 7.4). Activity of IDE was confirmed using insulin (Sigma-Aldrich) or the fluorogenic peptide Mca-RPPGFSAFK(Dnp)-OH (R&D Systems) as substrates.

Krasinski C.A., Zheng Q., Ivancic V.A., Spratt D.E, & Lazo N.D. (2018). The Longest Amyloid-β Precursor Protein Intracellular Domain Produced with Aβ42 Forms β-Sheet-Containing Monomers That Self-Assemble and Are Proteolyzed by Insulin-Degrading Enzyme. ACS chemical neuroscience, 9(12), 2892-2897.

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Enzymatic Activity Assay for IDE and NEP

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10 μg of cell or brain lysates were mixed with 10 μM of the fluorogenic peptide Mca-RPPGFSAFK(Dnp)-OH (R&D Systems) in reaction buffer (100 mM Tris-Cl, pH 7.5, 50 mM NaCl, and 10 mM ZnCl₂) and the fluorescent intensity of the cleaved fragments was monitored during 30 min at 37 °C with excitation at 320 nm and emission at 405 nm. Insulin (10 μM, Thermo Fisher Sci., RP-10908), a competitive substrate of IDE or thiorphan (40 μM, Santa Cruz Biotech., sc-201287), a potent inhibitor of NEP, was added to distinguish the specific enzyme activities. In both cases, we first determined the dose that achieves maximum substrate cleavage inhibition using 10 μg of lysate (Supplementary Fig. S1). IDE activity was defined as the activity sensitive to insulin inhibition and NEP activity was defined as the activity sensitive to thiorphan inhibition.

de Dios C., Bartolessis I., Roca-Agujetas V., Barbero-Camps E., Mari M., Morales A, & Colell A. (2019). Oxidative inactivation of amyloid beta-degrading proteases by cholesterol-enhanced mitochondrial stress. Redox Biology, 26, 101283.

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Quantitative SARS-CoV-2 RBD Binding Assay

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Recombinant proteins of human ACE2 extracellular enzymatic domain were purchased from R&D systems(933-ZN) and Sinobiological, Inc(10108-H08H). Recombinant proteins of SARS-CoV RBD (40150-V08B2) and SARS-CoV-2 RBD proteins (40592-V08H) were obtained from Sinobiological, Inc. Fluorogenic peptide substrates were obtained from R&D systems, where Mca-YVADAPK(Dnp)-OH (ES007) was used as an angiotensin II like substrate and Mca-RPPGFSAFK(Dnp)-OH(ES005) was a fluorogenic peptide derivative of Bradykinin according to manufacturer’s instruction. Non-fluorogenic bradykinin(BK, RPPGFSPFR), des-Arg⁹-bradykinin (desBK, RPPGFSPF) and angiotensin II (Ang II, DRVYIHPF) peptides were purchased from Genscript, Inc. Fluorogenic peptide Mca-Pro-Leu-OH (Bachem, M-1975) was used as calibration standard to obtain the molarity (pmol) to relative fluorescence unit (RFU) conversion factor on Synergy H1 fluorescent plate reader.

Lu J, & Sun P.D. (2020). High affinity binding of SARS-CoV-2 spike protein enhances ACE2 carboxypeptidase activity. bioRxiv.

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