A step-by-step protocol for assaying protein carbonylation in biological samples
| Autor: | Ranieri Rossi, Isabella Dalle-Donne, Maria Elisa Garavaglia, Aldo Milzani, Daniela Giustarini, Graziano Colombo, Marco Clerici |
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| Rok vydání: | 2016 |
| Předmět: |
Electrophoresis
0301 basic medicine Protein Carbonylation Blotting Western Clinical Biochemistry 2 4-Dinitrophenylhydrazine Biotin hydrazide Aldehyde-reactive probe Biotin-hydrazide Fluorescein-5-thiosemicarbazide Protein carbonylation Electrophoresis Gel Two-Dimensional Mass Spectrometry Oxidation-Reduction Proteins Spectrophotometry Analytical Chemistry Biochemistry Cell Biology High-performance liquid chromatography Adduct 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine 4-Dinitrophenylhydrazine Derivatization Polyacrylamide gel electrophoresis Gel Chromatography Blotting Chemistry General Medicine 030104 developmental biology 030220 oncology & carcinogenesis Two-Dimensional Western Carbonylation |
| Zdroj: | Journal of Chromatography B. 1019:178-190 |
| ISSN: | 1570-0232 |
| DOI: | 10.1016/j.jchromb.2015.11.052 |
| Popis: | Protein carbonylation represents the most frequent and usually irreversible oxidative modification affecting proteins. This modification is chemically stable and this feature is particularly important for storage and detection of carbonylated proteins. Many biochemical and analytical methods have been developed during the last thirty years to assay protein carbonylation. The most successful method consists on protein carbonyl (PCO) derivatization with 2,4-dinitrophenylhydrazine (DNPH) and consequent spectrophotometric assay. This assay allows a global quantification of PCO content due to the ability of DNPH to react with carbonyl giving rise to an adduct able to absorb at 366 nm. Similar approaches were also developed employing chromatographic separation, in particular HPLC, and parallel detection of absorbing adducts. Subsequently, immunological techniques, such as Western immunoblot or ELISA, have been developed leading to an increase of sensitivity in protein carbonylation detection. Currently, they are widely employed to evaluate change in total protein carbonylation and eventually to highlight the specific proteins undergoing selective oxidation. In the last decade, many mass spectrometry (MS) approaches have been developed for the identification of the carbonylated proteins and the relative amino acid residues modified to carbonyl derivatives. Although these MS methods are much more focused and detailed due to their ability to identify the amino acid residues undergoing carbonylation, they still require too expensive equipments and, therefore, are limited in distribution. In this protocol paper, we summarise and comment on the most diffuse protocols that a standard laboratory can employ to assess protein carbonylation; in particular, we describe step-by-step the different protocols, adding suggestions coming from our on-bench experience. |
| Databáze: | OpenAIRE |
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