The content of protein carbonyls was assessed as a measure of oxidative stress by the sensitive dot-blot assay as described. The lower limit of the detection was 2 pmol of carbonyls per microgram of protein. The assay was calibrated by dilutions of different proportions of oxidized and reduced bovine serum albumin (BSA) [Paesel-Lorei; Hanau, Germany]. BSA was oxidized in vitro by the Fenton reaction and completely reduced by NaBH4. The concentration of protein carbonyls in these BSA mixtures was quantified in a colorimetric assay.
Two-dimensional sodium dodecylsulfate-polyacrylamide gel electrophoresis was performed on two-dimensional gels (Nu-PAGE; Invitrogen; Carlsbad, CA) with modification for protein carbonyl identification. canadian healthcare mall
Briefly, following sample rehydration and isoelectric focusing on the immobilized pH gradient strips (pH 3 to 10; 7 cm; Immobiline; Amersham Biosciences, Uppsala, Sweden) for 2,000 Volt-hours. Samples were placed in 15-mL test tubes and incubated for 15 min in 2 N HCl with 10 mmol/L dinitrophenylhydrazin (DNPH) [Sigma; Taufkirchen, Germany] at 25°C. After the reaction, the samples were washed with 2 mol/L Tris-Base/30% glycerol (Plusone; Uppsala, Sweden) for 15 min. Molecular weight separation was performed on 4 to 12% Bis-Tris gels (Invitrogen). Subsequent proteins were transferred to the polyvinylidene fluoride membrane (Millipore; Bedford, MA) and analyzed with anti-2,4-dmitrophenyl antibodies (Sigma) for protein carbonyls.
Data are given as median values and range. Two groups were compared by Mann-Whitney U test, and three groups were compared by Kruskall-Wallis test with a Dunn post hoc test. The relation values between the different groups are given as a Spearman rank-sum correlation; p < 0.05 was considered significant.