These problems were obviated by employing dimethyl sulfoxide (DMSO) as solvent. Few proteins are soluble in HCl, which will not wet PVDF membranes. It has limited, but sufficient, solubility in strong acids and that is the reason stock solutions are usually made in 2M HCl. Our goal was to develop a more sensitive dot blot method in which many samples of ~ 1 μl volume could be applied to one PVDF membrane, allowing convenient quantitation of multiple samples with replicates.ĭNPH has limited solubility in water and many common solvents. Still, in our hands, 10 to 20 μg protein per sample is needed to obtain duplicate analyses. When detected with anti-DNPH antibodies carrying fluorescent labels, the Western blot is quantitative and fairly sensitive. Immunochemical detection is now more commonly utilized, either as an ELISA or as a Western blot of SDS-PAGE separated proteins. Spectrophotometry was initially used for quantitation of derivatized proteins. The most commonly employed is the classical carbonyl reagent, 2,4-dinitrophenylhydrazine (DNPH) 1, perhaps first applied to proteins by Dixon. A variety of derivatizing reagents have been utilized for spectrophotometric, fluorimetric, and immunochemical analysis.
In this context, the term protein carbonylation refers to “reactive” carbonyl groups, that is, to aldehydes and ketones.ĭetection and quantitation of carbonylated proteins is accomplished after derivatization of the carbonyl groups. The National Library of Medicine’s Medical Subject Headings entry for protein carbonylation notes that, “It is a standard marker for oxidative stress.”. Protein carbonylation occurs in many of these modifications, providing an integrated assessment of oxidative damage. A large number of the resulting modifications of proteins have been characterized and studied. As a consequence of the oxidative stress, cells carry an increased burden of oxidatively damaged macromolecules, including nucleic acids, lipids, and proteins. The list of diseases and processes in which oxidative damage is implicated reads like a textbook of pathology and includes atherosclerosis, cancer, neurodegenerative diseases such as Alzheimers and Parkinsons, and the aging process. It also occurs as a consequence of acute or chronic oxidative stress in many conditions. Oxidative modification of proteins, both reversible and irreversible, occurs during redox signaling and other cellular processes.
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