Development of immunoblotting techniques for DNA radical detection.

Abstract

Radical damage to DNA has been implicated in cell death, cellular dysfunction, and cancer. A recently developed method for detecting DNA radicals uses the nitrone spin trap DMPO (5,5-dimethyl-1-pyrroline N-oxide) to trap radicals. The trapped radicals then decay into stable nitrone adducts detectable with anti-DMPO antibodies and quantifiable by ELISA or dot-blot assay. However, the sequences of DNA that are damaged are likely to be as important as the total level of damage. Therefore, we have developed immunoblotting methods for detection of DNA nitrone adducts on electrophoretically separated DNA, comparable to Western blotting for proteins. These new techniques not only allow the assessment of relative radical adduct levels, but can reveal specific DNA fragments, and ultimately nucleotides, as radical targets. Moreover, we have determined that denaturation of samples into single-stranded DNA enhances the detection of DNA-DMPO adducts in our new blotting methods and also in ELISA.

• Authors: Fiona A Summers, Ronald P Mason, Marilyn Ehrenshaft
• Journal: Free radical biology & medicine (Free Radic Biol Med) Vol. 56 Pg. 64-71 (Mar 2013) ISSN: 1873-4596 [Electronic] United States
• PMID: 23142572 (Publication Type: Journal Article, Research Support, N.I.H., Intramural)
• Copyright: Published by Elsevier Inc.

Chemical References

• Cyclic N-Oxides
• Free Radicals
• 5,5-dimethyl-1-pyrroline-1-oxide
• DNA

Topics

• Cyclic N-Oxides (chemistry)
• DNA (analysis)
• Electrophoresis, Agar Gel
• Enzyme-Linked Immunosorbent Assay
• Free Radicals (analysis)
• Immunoblotting (methods)
• Molecular Structure