Decreased sensitivity associated with an altered formulation of a commercially available kit for detection of protein carbonyls

Carbonylation is a commonly studied form of oxidative modification to proteins which can be conveniently detected using commercially available kits. The most common of these kits is the OxyblotTM Protein Oxidation Detection Kit (Chemicon/Millipore). Over the past year we have observed severely diminished sensitivity of these kits which was shown to be a result of a change in the formulation of one of the components supplied in the kit. This component, the 10X 2,4-dinitrophenylhydrazine derivatization solution, which had previously been dissolved in 100% trifluoroacetic acid (TFA), was now dissolved in 2N hydrochloric acid, which according to our results is not acid enough. Further, we observed that upon storage even DNPH dissolved in TFA is subject to degradation. Based on these studies, we make recomendations that should improve the sensitivity and reproducibilty of this assay.