Analysis of oxidative stress-induced protein carbonylation using fluorescent hydrazides

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Data de publicació
2012Autor/a
Hoogh, Anouk de
Alsina Obiols, David
Citació recomanada
Tamarit Sumalla, Jordi;
Hoogh, Anouk de;
Obis Monné, Èlia;
Alsina Obiols, David;
Cabiscol Català, Elisa;
Ros Salvador, Joaquim;
.
(2012)
.
Analysis of oxidative stress-induced protein carbonylation using fluorescent hydrazides.
Journal of Proteomics, 2012, vol. 75, núm. 12, p. 3778-3788.
https://doi.org/10.1016/j.jprot.2012.04.046.
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Protein carbonyl detection has been commonly used to analyze the degree of damage to
proteins under oxidative stress conditions. Most laboratories rely on derivatization of
carbonyl groups with dinitrophenylhydrazine followed by Western blot analysis using
antibodies against the dinitrophenyl moiety. This paper describes a protein carbonyl
detection method based on fluorescent Bodipy, Cy3 and Cy5 hydrazides. Using this
approach, Western blot and immunodetection are no longer needed, shortening the
procedure and increasing accuracy. Combination of Cy3 and Cy5 hydrazides allows
multiplexing analyses in a single two-dimensional gel. Derivatization with Bodipy
hydrazide allows easy matching of the spots of interest and those obtained by general
fluorescent protein staining methods, which facilitates excising target proteins from the
gels and identifying them. This method is effective for detecting protein carbonylation in
samples of proteins submitted to metal-catalyzed oxidation “in vitro” and assessing the
effect of hydrogen peroxide and chronological aging on protein oxidative damage in yeast
cells.