Figure 3.

Immuno-spin trapping analysis of DNA radicals. (a) We mixed the following components: 20 µM DNA (as nucleotides), 20 µM Cu²⁺ (as chloride salt), 20 µM H₂O₂ and/or 50 mM DMPO in 100 mM chelexed PB (pH 7.4), in a total volume of 300 µl. We added a 10-X stock DNA solution (30 µl) and the other components from a 100-X stock solution (3 µl). After 1 h incubation, we stopped the reaction with 3 µl of 1 M KCN or 100 mM DTPA. Stopping the reaction with either reagent gave similar results. After stopping, we froze the samples until analysis. (b) The procedure was as described for (a), but we added different concentrations of Cu²⁺ or omitted one of the components in the reaction mixture. *P < 0.05 with respect to “zero” Cu²⁺. (c) The procedure was as described for (b), but we varied the final concentration of H₂O₂ in the reaction mixtures. *P <0.05 (with respect to “zero” H₂O₂. (d) We added 20 µM DNA, 50 mM DMPO, and equal concentrations of Cu²⁺ and H₂O₂ (e.g., “5” corresponds to 5 µM Cu²⁺ and 5 µM H₂O₂, and so on). *P < 0.05 with respect to “5”(e.g., 5 µM Cu²⁺:5 µM H₂O₂). (e) The procedure was as described for (c), but we added different final concentrations of DMPO. (f) We mixed 20 µM DNA, 20 µM Cu²⁺, 50 mM DMPO and 20 µM H₂O₂, and then added 3 µl of 1 M KCN to stop the reaction at different times after the addition of H₂O₂. For time “zero”, we added KCN just before H₂O₂. We performed the immuno-spin trapping analysis as described in the PROCEDURE. Insets show the immuno-slot blot corresponding to the samples analyzed by ELISA in the main graph. We compared AP (blue bars) with our improved developing system using HRP (red bars). We developed the immuno-slot blot using the HRP system. Data show the mean ± s.e.m. from three separate experiments in quadruplicate (n = 12).