Immuno-spin trapping analyses of DNA radicals.

Abstract

Immuno-spin trapping is a highly sensitive method for detecting DNA radicals in biological systems. This technique involves three main steps: (i) in situ and real-time trapping of DNA radicals with the nitrone spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), thus forming DMPO-DNA nitrone adducts (referred to here as nitrone adducts); (ii) purification of nitrone adducts; and (iii) analysis of nitrone adducts by heterogeneous immunoassays using Abs against DMPO. In experiments, DMPO is added prior to the formation of free radicals. It diffuses easily through all cell compartments and is present when DNA free radicals are formed as a result of oxidative damage. Due to its low toxicity, DMPO can be used in cells at high enough concentrations to out-compete the normal reactions of DNA radicals, thus ensuring a high yield of DNA nitrone adducts. Because both protein and DNA nitrone adducts are formed, it is important that the DNA be pure in order to avoid misinterpretations. Depending on the model under study, this protocol can be completed in as few as 6 h.