Reduction of the nitro group during sample preparation may cause underestimation of the nitration level in 3-nitrotyrosine immunoblotting.
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.
Söderling
Ann-Sofi
AS
Sahlgrenska Academy, Department of Clinical Physiology, Sahlgrenska University Hospital, Göteborg, Sweden.
Hultman
Lena
L
Delbro
Dick
D
Højrup
Peter
P
Caidahl
Kenneth
K
D053000:Analytic Sample Preparation Methods; D000804:Angiotensin II / Q000737:chemistry; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002851:Chromatography, High Pressure Liquid; D015151:Immunoblotting; D007150:Immunohistochemistry; D008099:Liver / Q000166:cytology; D008168:Lung / Q000166:cytology; D010084:Oxidation-Reduction; D051381:Rats; D012709:Serum Albumin / Q000302:isolation & purification; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D012709:Serum Albumin; D000804:Angiotensin II; C002744:3-nitrotyrosine; D014443:Tyrosine