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Reduction of the ni...
Reduction of the nitro group during sample preparation may cause underestimation of the nitration level in 3-nitrotyrosine immunoblotting
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Söderling, A. S. (författare)
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Hultman, L. (författare)
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- Delbro, Dick, 1950 (författare)
- Gothenburg University,Göteborgs universitet,Institutionen för kliniska vetenskaper,Institute of Clinical Sciences
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Hojrup, P. (författare)
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- Caidahl, Kenneth, 1949 (författare)
- Karolinska Institutet,Gothenburg University,Göteborgs universitet,Institutionen för medicin, avdelningen för molekylär och klinisk medicin,Institute of Medicine, Department of Molecular and Clinical Medicine
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(creator_code:org_t)
- Elsevier BV, 2007
- 2007
- Engelska.
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Ingår i: Journal of chromatography. - : Elsevier BV. - 1570-0232. ; 851:1-2, s. 277-86
- Relaterad länk:
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https://gup.ub.gu.se...
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https://doi.org/10.1...
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Abstract
Ämnesord
Stäng
- 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.
Nyckelord
- *Analytic Sample Preparation Methods
- Angiotensin II/chemistry
- Animals
- Antibodies
- Monoclonal/immunology
- Chromatography
- High Pressure Liquid
- Immunoblotting
- Immunohistochemistry
- Liver/cytology
- Lung/cytology
- Oxidation-Reduction
- Rats
- Serum Albumin/isolation & purification
- Spectrometry
- Mass
- Electrospray Ionization
- Spectrometry
- Mass
- Matrix-Assisted Laser Desorption-Ionization
- Tyrosine/*analogs & derivatives/metabolism
Publikations- och innehållstyp
- ref (ämneskategori)
- art (ämneskategori)
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