@article{06b3b7b0d94e11dc860c000ea68e967b,
title = "Reduction of the nitro group during sample preparation may cause underestimation of the nitration level in 3-nitrotyrosine immunoblotting",
abstract = "We noted differences in the antibody response to 3-nitrotyrosine (NO(2)Tyr) in fixed and non-fixed tissues, and studied therefore potential problems associated with non-fixed tissues in Western blot analyses. Three different monoclonal anti-nitrotyrosine antibodies in Western blot analysis of inflammatory stimulated rat abdominal, liver and lung tissue homogenates caused no immunoreactivity, in contrast to a polyclonal nitrotyrosine antibody applied in fixed and non-fixed tissues. Western blot studies using both mono- and polyclonal antibodies showed a temperature- and heme group-dependent reduction of NO(2)Tyr in nitrated rat and bovine serum albumin incubated with dithiothreitol. Mass spectrometric analyses of a nitrated peptide angiotensin II revealed under similar conditions a positive temperature effect between 56 and 70 degrees C on reduction of NO(2)Tyr to 3-aminotyrosine which is not detected by anti-NO(2)Tyr antibodies. Western blot analysis may therefore underestimate the level of tissue nitration, and factors causing a reduction of NO(2)Tyr during sample preparation might conceal the actual nitration of proteins.",
keywords = "Analytic Sample Preparation Methods, Angiotensin II, Animals, Antibodies, Monoclonal, Chromatography, High Pressure Liquid, Immunoblotting, Immunohistochemistry, Liver, Lung, Oxidation-Reduction, Rats, Serum Albumin, Spectrometry, Mass, Electrospray Ionization, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Tyrosine",
author = "Ann-Sofi S{\"o}derling and Lena Hultman and Dick Delbro and Peter H{\o}jrup and Kenneth Caidahl",
year = "2007",
month = may,
day = "15",
doi = "10.1016/j.jchromb.2007.02.036",
language = "English",
volume = "851",
pages = "277--286",
journal = "Journal of Chromatography B",
issn = "1570-0232",
publisher = "Elsevier",
number = "1-2",
}