Absolute quantity of MAO-B in platelet-rich plasma was determined using a sensitive ELISA developed in our laboratory. Levels of MAO isoform A (MAO-A) in platelets and blood are undetectable with standard ELISA, so this isoform was not measured in this study. A standard curve of known concentrations of MAO-B was generated with recombinant MAO-B (Sigma-Aldrich, St. Louis, Missouri) and incubated on Immulon-coated 96-well assay plates (Thermo Fisher Scientific; Waltham, Massachusetts) along with diluted plasma samples. Samples were incubated overnight at 4°C and subsequently washed with a solution of phosphate-buffered saline (PBS) plus 0.05% Tween-20 and blocked for 2 hours with 5% bovine serum albumin at 37°C. Samples were then incubated with primary antibody for MAO-B (Abcam, Cambridge, England) for 2 h at 37°C (or overnight at 4°C). Following another wash with PBS plus 0.05% Tween-20, samples were incubated with horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, Inc.; Dallas, Texas) for 2 h at 37°C. Following an additional wash, samples were incubated with Amplex Red (10 mM; Thermo Fisher, Waltham, Massachusetts). Concentrations of MAO-B enzyme within each sample were determined by fitting to the standard curve of recombinant MAO-B within each plate and normalized to plasma volume.
Recombinant mao b
Manufactured by Merck Group
Recombinant MAO-B is a laboratory product used for research purposes. It is a recombinant form of the enzyme monoamine oxidase B (MAO-B), which is involved in the metabolism of neurotransmitters and other biogenic amines. The product is intended for use in research studies and experiments, but its specific applications and intended use should not be extrapolated or interpreted beyond the core function described.
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2 protocols using recombinant mao b
1
Quantification of Platelet MAO-B Levels
2
Fluorometric Assay for hMAO B Inhibition
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Inhibition of hMAO B was evaluated using fluorometric Monoamine Oxidase assay as described by Łażewska et al. [24 (link)]. Recombinant MAO B was purchased from Sigma Aldrich (M7441). As the references were used safinamide (1 μM; Sigma Aldrich) and pargyline (10 µM; Sigma Aldrich). The assay was carried out in 96-well plate. Tested compounds dissolved in DMSO (1 μM) were added to wells that contained 98 µL of enzyme dilution (1 µg/well) in phosphate buffer (50 mM, pH 7.4). After the 30 min of preincubation at room temperature AmplexTM Red (50 µL) and horse radish peroxidase (4 U/mL) were added. An enzymatic reaction started following addition of para-tyramine solution (200 μM). The signal was measured after 1h (excitation at 570 nm and emission at 585 nm) using EnSpire® multimode plate reader (Perkin Elmer, Inc.). Compounds that inhibited the enzyme by more than 50% of pargyline were chosen for IC50 evaluation. Each experiment was performed in duplicate.
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