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Methods Mol Biol. 2009;536:457-62. doi: 10.1007/978-1-59745-542-8_46.

Detection of protein carbonyls by means of biotin hydrazide-streptavidin affinity methods.

Author information

1
Free Radical Biology and Aging Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK, 73104, USA. Kenneth-Hensley@omrf.ouhsc.edu

Abstract

Oxidative posttranslational protein modifications occur as a normal process of cell biology and to a greater extent during pathogenic conditions. The detection and quantitation of protein oxidation has posed a continuing challenge to bioanalytical chemists because the products of oxidative protein damage are chemically diverse, protein oxidation generally occurs at low background levels, and the complexity of biological samples introduces high background noise when standard techniques such as immunolabeling are applied to "dirty" tissue extracts. A refinement of classic reductive amination methods has been developed, which circumvents these difficulties by incorporating a biotin label at sites of protein carbonylation. Biotin hydrazide-labeled proteins are detectable using standard streptavidin-coupled detection techniques such as peroxidase-catalyzed chemiluminescence of immunoblots. Advantages of the biotin hydrazide-labeling technique are its sensitivity and its lack of reliance upon antibodies that inevitably suffer from nonspecific background noise and contaminating endogenous immunoglobulins.

PMID:
19378083
DOI:
10.1007/978-1-59745-542-8_46
[Indexed for MEDLINE]

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