Analysis of oxidative stress-induced protein carbonylation using fluorescent hydrazides
Hoogh, Anouk de
Alsina Obiols, David
MetadataShow full item record
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.
Is part ofJournal of Proteomics, 2012, vol. 75, núm. 12, p. 3778-3788
European research projects
Showing items related by title, author, creator and subject.
Moreno Cermeño, Armando J.; Alsina Obiols, David; Cabiscol Català, Elisa; Tamarit Sumalla, Jordi; Ros Salvador, Joaquim (Elsevier, 2013)Frataxin is a mitochondrial protein involved in iron metabolism whose deficiency in humans causes Friedreich ataxia. We performed transcriptomic and proteomic analyses of conditional Yeast Frataxin Homologue (Yfh1) mutants ...
Major targets of iron-induced protein oxidative damage in frataxin-deficient yeasts are magnesium-binding proteins Irazusta, Verónica Patricia; Moreno Cermeño, Armando J.; Cabiscol Català, Elisa; Ros Salvador, Joaquim; Tamarit Sumalla, Jordi (Elsevier, 2008)Iron accumulation has been associated with several pathological conditions such as Friedreich ataxia. This human disorder is caused by decreased expression of frataxin. Iron-overload triggers oxidative stress, but the ...
Yeast frataxin mutants display decreased superoxide dismutase activity crucial to promote protein oxidative damage Irazusta, Verónica Patricia; Obis Monné, Èlia; Moreno Cermeño, Armando J.; Cabiscol Català, Elisa; Ros Salvador, Joaquim; Tamarit Sumalla, Jordi (Elsevier, 2010)Iron overload is involved in several pathological conditions, including Friedreich ataxia, a disease caused by decreased expression of the mitochondrial protein frataxin. In a previous study, we identified 14 proteins ...