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Fluorogenic tagging of protein 3-nitrotyrosine with 4-(aminomethyl)benzenesulfonate (ABS) in tissues: a useful alternative to immunohistochemistry for fluorescence microscopy imaging of protein nitration
Sharov, Victor S. Pal, Ranu Dremina, Elena S. Michaelis, Elias K. Schoneich, Christian
Sharov, Victor S.
Pal, Ranu
Dremina, Elena S.
Michaelis, Elias K.
Schoneich, Christian
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Abstract
Protein tyrosine nitration is a common biomarker of biological aging and diverse pathologies associated with the excessive formation of reactive oxygen and nitrogen species. Recently, we suggested a novel fluorogenic derivatization procedure for the detection of 3-nitrotyrosine (3-NT) using benzylamine derivatives to convert specifically protein or peptide bound 3-NT to a highly fluorescent benzoxazole product. In the current study, we applied this procedure to fluorogenic derivatization of protein 3-NT in sections from adult rat cerebellum in order to: (i) test this method in imaging nitrated proteins in fixed brain tissue sections, and (ii) compare the chemical approach to immunohistochemical labeling with anti-3-NT antibodies. Immunofluorescence analysis of cerebellar sections using anti-3-NT antibodies showed differential levels of immunostaining in the molecular, Purkinje, and granule cell layers of the cerebellar cortex; in agreement with previous reports, the Purkinje cells were most highly labeled. Importantly, fluorogenic derivatization reactions of cerebellar proteins with 4-(aminomethyl)benzenesulfonic acid (ABS) and K3Fe(CN)6 at pH 9, following sodium dithionite (SDT) reduction of 3-NT to 3-aminotyrosine (3-AT), showed a very similar pattern of relative intensity of cell labeling and improved resolution when compared with antibody labeling. Our data demonstrate that ABS-derivatization may be either a useful alternative or a complimentary approach to immunolabeling in imaging protein nitration in cells and tissues, including under conditions of dual labeling with antibodies to cell proteins, thus allowing for cellular co-localization of nitrated proteins and any protein of interest.
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Date
2013-11-15
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Elsevier
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Keywords
Protein nitration, 3-nitrotyrosine, Cerebellum, Purkinje cells, 4-(aminomethyl)benzenesulfonate (ABS), Fluorogenic derivatization, Fluorescence microscopy, Immunohistochemistry, Synaptophysin, SIN-1
Citation
Sharov, V.s., R. Pal, E.s. Dremina, E.k. Michaelis, and C. Schoneich. "Fluorogenic Tagging of Protein 3-nitrotyrosine with 4-(aminomethyl) benzene Sulfonate in Tissues: A Useful Alternative to Immunohistochemistry for Fluorescence Microscopy Imaging of Protein Nitration." Free Radical Biology and Medicine 53.10 (2012): 1877-885.