Immunological detection and quantification of oxidized proteins by labelling with digoxigenin.
An immunological assay, based on the digoxigenin/anti-digoxigenin system, was developed to detect and quantify carbonyl moieties that result from oxidative damage to proteins. Bovine serum albumin (BSA) was oxidized by a hydroxyl radical-generating system consisting of ascorbate/Fe(III)/O2. The resulting albumin-derived carbonyls were labelled with digoxigenin-hydrazide and detected by dot blotting with an anti-digoxigenin antibody conjugated to alkaline phosphatase. Quantification was carried out by a densitometric analysis. This system allows the detection of a pmole-amount of carbonyl groups on blots. The assay covers a range of sensitivity from 1.26 to 126 pmoles. Another feature of this method is its application to a complex protein mixture (homogenate) to analyze the oxidative status of individual proteins, as are shown for intestinal brush border membrane homogenate of a rat.
Bautista
J
J
Department of Biochemistry, Bromatology and Toxicology, Faculty of Pharmacy, University of Sevilla, Spain. jdbaut@cica.es
Mateos-Nevado
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MD
D000818:Animals; D000906:Antibodies; D002417:Cattle; D004076:Digoxigenin; D015151:Immunoblotting / Q000379:methods; D010084:Oxidation-Reduction; D011506:Proteins / Q000032:analysis*; D051381:Rats; D017208:Rats, Wistar; D012710:Serum Albumin, Bovine / Q000032:analysis
D000906:Antibodies; D011506:Proteins; D012710:Serum Albumin, Bovine; D004076:Digoxigenin