The determination of MAO-A and MAO-B inhibition was performed using commercially available recombinant human MAO-A and MAO-B enzymes expressed in baculovirus-infected insects sells (Sigma-Aldrich, M7316 and M7441) applying the commercially available Amplex® Red monoamine oxidase assay kit (Invitrogen A12214). The assays were performed as previously described. The determination of rat MAO-B inhibition was performed using mitochondrial-enriched fractions from male Sprague Dawley rat livers. The assays were conducted as previously described (Stössel et al., 2013 (link)).
M7316
Manufactured by Merck Group
Sourced in United States
The M7316 is a laboratory equipment product from Merck Group. It serves as a core function for specific laboratory applications. No further details on its intended use can be provided in an unbiased and factual manner.
Automatically generated - may contain errors
Lab products found in correlation
Amplex red monoamine oxidase assay kit, by Thermo Fisher Scientific (1 mentions)
Pargyline, by Merck Group (1 mentions)
Kynuramine, by Merck Group (1 mentions)
Spectramax m2, by Molecular Devices (1 mentions)
P tyramine, by Merck Group (1 mentions)
Hmao a, by Merck Group (1 mentions)
Amplex red, by Thermo Fisher Scientific (1 mentions)
Mao b, by Merck Group (1 mentions)
Lsd1 fluorometric drug discovery kit, by Enzo Life Sciences (1 mentions)
5 protocols using m7316
1
Determination of MAO Enzyme Inhibition
2
Quantitative MAO Inhibitor Assay
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MAO-A
and MAO-B inhibitory
activities were assayed using the method in our previous report with
slight modification.16 (link) Human recombinant
MAO-A solution (3 μL, M7316, Sigma-Aldrich, St. Louis, MO) or
7 μL of MAO-B solution (M7441, Sigma-Aldrich) was diluted with
1100 μL of potassium phosphate buffer (0.1 M, pH 7.4). Potassium
phosphate buffer (140 μL), 8 μL of kynuramine (final concentration
is 30 μM, Sigma-Aldrich) in potassium phosphate buffer, and
2 μL of a dimethyl sulfoxide (DMSO) inhibitor solution [final
DMSO concentration of 1% (v/v)] were mixed and preincubated at 37
°C for 10 min. Diluted MAO-A or MAO-B solution (50 μL)
was then added to each well. The reaction mixture was further incubated
at 37 °C, and the reaction was stopped after 20 min by the addition
of 75 μL of 2 M NaOH. The product generated by MAO-A or MAO-B,
4-quinolinol, is fluorescent and was measured at Ex 310 nm/Em 400
nm using a microplate reader (SPECTRA MAX M2, Molecular Devices, Tokyo,
Japan). DMSO without the test compound was used as the negative control,
and pargyline (Sigma-Aldrich) was used as a positive control.17 (link) The IC50 values were estimated using
Prism software (version 5.02; GraphPad, San Diego, CA).
and MAO-B inhibitory
activities were assayed using the method in our previous report with
slight modification.16 (link) Human recombinant
MAO-A solution (3 μL, M7316, Sigma-Aldrich, St. Louis, MO) or
7 μL of MAO-B solution (M7441, Sigma-Aldrich) was diluted with
1100 μL of potassium phosphate buffer (0.1 M, pH 7.4). Potassium
phosphate buffer (140 μL), 8 μL of kynuramine (final concentration
is 30 μM, Sigma-Aldrich) in potassium phosphate buffer, and
2 μL of a dimethyl sulfoxide (DMSO) inhibitor solution [final
DMSO concentration of 1% (v/v)] were mixed and preincubated at 37
°C for 10 min. Diluted MAO-A or MAO-B solution (50 μL)
was then added to each well. The reaction mixture was further incubated
at 37 °C, and the reaction was stopped after 20 min by the addition
of 75 μL of 2 M NaOH. The product generated by MAO-A or MAO-B,
4-quinolinol, is fluorescent and was measured at Ex 310 nm/Em 400
nm using a microplate reader (SPECTRA MAX M2, Molecular Devices, Tokyo,
Japan). DMSO without the test compound was used as the negative control,
and pargyline (Sigma-Aldrich) was used as a positive control.17 (link) The IC50 values were estimated using
Prism software (version 5.02; GraphPad, San Diego, CA).
3
Inhibitory Activity of Compounds on MAOs
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The inhibitory activities of the compounds 3a-i and 3e1-e11 towards MAOs were assayed by the Amplex Red fluorescence method. The recombinant human MAOs (hMAO-A, M7316 and hMAO-B, M7441) and p-tyramine (T90344) were obtained from Sigma-Aldrich (St. Louis, MO, USA). hMAO-A and hMAO-B and Amplex Red assay kit were used to determine the production of H2O2 from substrate p-tyramine. The test compounds were dissolved in DMSO and then diluted to different concentrations with PBS buffer solution (DMSO <0.01%). 80 μL of hMAO-A or hMAO-B and 20 μL of different concentrations of compounds were added to a 96-well black microtiter plate, and then incubated for 15 min at 37°C in the dark. After this period, a substrate mixture was added quickly and the results were tested by a multi-detection microplate fluorescence reader with excitation/emission wavelengths of 544/590 nm. Data were shown as mean±SD of three independent experiments.
4
Kinetic Analysis of LSD1 Inhibition
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The kinetics of LSD1 inhibition by bomedemstat were analyzed using the LSD1 Fluorometric Drug Discovery Kit (Enzo Life Sciences #BML-AK544) according to the manufacturer's directions with LSD1 [32 nM] and bomedemstat [5 nM-6.944 μM]. All substrates and peptides were mixed simultaneously with measurements commencing immediately and taken every 30 s for 30 min. Reaction rates (Δ[product]/time) were calculated for each 30 s interval using 2.5 min rolling averages, which were used to calculate IC 50 values for each 30 s time interval via GraphPad Prism (nonlinear regression curve fit, four parameters).
To calculate single-timepoint IC 50 values, bomedemstat (0.5 nM-100 μM) was preincubated with LSD1 [50 nM, RBC #PDM-11-350], MAO-A [0.5 U/mL; Sigma-Aldrich #M7316], or MAO-B [1 U/mL; Sigma-Aldrich #M7441] for 30 min before adding HRP [0.2 U/mL; Sigma-Aldrich #P8375] and substrates (Amplex Red [20 μM; Invitrogen #Aa36006] and either H3(1-21)K4Me2 [10 μM] peptide for LSD1 or tyramine [10 μM] for MAO-A/B). The reaction buffer included 50 mM Tris-HCl pH 7.5, 0.05% CHAPS, and 1% DMSO in dH 2 O. IC 50 values were calculated from five replicate experiments using GraphPad Prism (nonlinear regression curve fit, four parameters).
To calculate single-timepoint IC 50 values, bomedemstat (0.5 nM-100 μM) was preincubated with LSD1 [50 nM, RBC #PDM-11-350], MAO-A [0.5 U/mL; Sigma-Aldrich #M7316], or MAO-B [1 U/mL; Sigma-Aldrich #M7441] for 30 min before adding HRP [0.2 U/mL; Sigma-Aldrich #P8375] and substrates (Amplex Red [20 μM; Invitrogen #Aa36006] and either H3(1-21)K4Me2 [10 μM] peptide for LSD1 or tyramine [10 μM] for MAO-A/B). The reaction buffer included 50 mM Tris-HCl pH 7.5, 0.05% CHAPS, and 1% DMSO in dH 2 O. IC 50 values were calculated from five replicate experiments using GraphPad Prism (nonlinear regression curve fit, four parameters).
5
Quantification of Oxidative Stress Biomarkers
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