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 Oxyblot 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.
Cardiac Metabolism Laboratory, Center for Heart and Lung Research, The Feinstein Institute for Medical Research, Manhasset NY, USA.
Ping
P
Wang
Saul R
SR
Powell
D000818:Animals; D001671:Biochemistry / Q000379:methods; D045424:Complex Mixtures / Q000737:chemistry*; D005449:Fluorenes / Q000737:chemistry*; D006801:Humans; D006834:Hydrazines / Q000737:chemistry*; D006851:Hydrochloric Acid / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D050050:Protein Carbonylation; D011933:Reagent Kits, Diagnostic; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D045424:Complex Mixtures; D005449:Fluorenes; D006834:Hydrazines; D011933:Reagent Kits, Diagnostic; C446799:dinitrophenylhydrazine; C512801:trifluorenylamine; D006851:Hydrochloric Acid