INVESTIGADORES
GOMEZ-MEJIBA sandra Esther
artículos
Título:
IMMUNO-SPIN TRAPPING ANALYSES OF DNA RADICALS
Autor/es:
RAMIREZ DC; SANDRA E GOMEZ-MEJIBA; MASON, RP
Revista:
NATURE PROTOCOLS
Editorial:
NATURE PUBLISHING GROUP
Referencias:
Lugar: Londres; Año: 2007 p. 512 - 522
ISSN:
1754-2189
Resumen:
mmuno-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.