Reduction of the nitro group during sample preparation may cause underestimation of the nitration level in 3-nitrotyrosine immunoblotting.
J Chromatogr B Analyt Technol Biomed Life Sci
; 851(1-2): 277-86, 2007 May 15.
Article
en En
| MEDLINE
| ID: mdl-17368119
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.
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Banco de datos:
MEDLINE
Asunto principal:
Tirosina
/
Métodos Analíticos de la Preparación de la Muestra
Límite:
Animals
Idioma:
En
Revista:
J Chromatogr B Analyt Technol Biomed Life Sci
Asunto de la revista:
ENGENHARIA BIOMEDICA
Año:
2007
Tipo del documento:
Article
País de afiliación:
Suecia