- #QUANTITATION OF PROTEIN CARBONYLATION BY DOT BLOT SERIES#
- #QUANTITATION OF PROTEIN CARBONYLATION BY DOT BLOT FREE#
Vásquez-Garzón, Verónica R Rouimi, Patrick Jouanin, Isabelle Waeg, Georg Zarkovic, Neven Villa-Treviño, Saul Guéraud, FrançoiseĪmong disruptions induced by oxidative stress, modifications of proteins, particularly irreversible carbonylation, are associated with the development of several diseases, including cardiovascular diseases, neurodegenerative diseases, and cancer. Then, we investigated a HPLC procedure which uses sodium dodecyl sulfate in the mobile phase but this was also found to be unsuitable for the reactive protein carbonyl assay in tissues.Įvaluation of three simple direct or indirect carbonyl detection methods for characterization of oxidative modifications of proteins. Nevertheless, on using the procedure described in the literature to measure total "reactive carbonyls" in rat liver and gerbil brain cortex, no change with age or PBN treatment was found.
#QUANTITATION OF PROTEIN CARBONYLATION BY DOT BLOT FREE#
The unwashed free DNPH was another major problem in the assay because of its very strong absorption around 370 nm, where reactive carbonyls were quantitated. This streptomycin sulfate treatment procedure, however, had no effect on the reactive carbonyl measurement of an oxidized protein sample. Using the streptomycin sulfate treatment combined with a dialysis step, we were successful in removing most nucleic acids from a crude tissue extract, but then the reactive carbonyl level in the crude tissue extract was too low to be reliably measured. The nucleic acids in tissues could be a major problem encountered in the assay. Our results indicate that reactive protein carbonyls in normal crude tissue extracts cannot be reliably measured by this method, although it does reliably measure reactive carbonyls in purified proteins which have been oxidatively modified in vitro. This method uses 2,4-dinitrophenylhydrazine (DNPH) and includes a washing procedure. Here we specifically study the method that was used to measure reactive protein carbonyls in tissues. This hypothesis is also supported by the report of a novel effect of N-tert-butyl-alpha-phenylnitrone (PBN) in reversing these age-dependent changes.
#QUANTITATION OF PROTEIN CARBONYLATION BY DOT BLOT SERIES#
This hypothesis is based on a series of reports showing an age-dependent increase in protein carbonyl content and an age-dependent loss of enzyme function. Difficulties in measuring reactive protein carbonyls in tissues using 2,4-dinitrophenylhydrazine.Ī current hypothesis explaining the aging process implicates the accumulation of oxidized protein in animal tissues. We present protocols for the derivatization and quantification of protein carbonylation with these two methods, including a newly described dot blot with greatly increased sensitivity. It is most often measured spectrophotometrically or immunochemically by derivatizing proteins with the classical carbonyl reagent 2,4 dinitrophenylhydrazine (DNPH). Protein carbonylation is the most commonly used measure of oxidative modification of proteins. The focus of this review is on the use of proteomics tools and methods to identify oxidized proteins along with specific sites of oxidative damage and the consequences of protein oxidation. The development of new selection and enrichment techniques coupled with advances in mass spectrometry are allowing identification of hundreds of new carbonylated protein products from a broad range of proteins located at many sites in biological systems. Studies of oxidative stress are complicated by the low concentration of oxidation products and wide array of routes by which proteins are carbonylated. Protein carbonyl groups can be generated directly (by amino acids oxidation and the a-amidation pathway) or indirectly by forming adducts with lipid peroxidation products or glycation and advanced glycation end-products. Carbonylation is an irreversible post translational modification (PTM) that often leads to the loss of protein function and can be a component of multiple diseases. PROTEOMIC IDENTIFICATION OF CARBONYLATED PROTEINS AND THEIR OXIDATION SITESĮxcessive oxidative stress leaves a protein carbonylation fingerprint in biological systems.
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