Oxidative dna damage elisa kit

The oxidative DNA damage ELISA kit (Cell Biolabs) is a competitive enzyme immunoassay available for rapid detection and quantification of 8-hydroxydeoxyguanosine (8-OHdG), a ubiquitous marker of oxidative stress and a by-product of oxidative DNA damage from cellular DNA samples^{53 (link)}. The quantity of 8-OHdG in an unknown sample is determined by comparing its absorption with that of a known standard curve. DNA was isolated from the treated and untreated samples using a bacterial DNA isolation kit (Promega) and equal amounts of DNA samples were analyzed for the detection of the 8‐OHdG level using the oxidative DNA damage ELISA kit.

Lipid peroxidation was determined by a method that measures the amount of thiobarbituric acid reactivity by the amount of malondialdehyde (MDA) formed during acid hydrolysis of the lipid peroxide compounds using the Lipid Peroxidation MDA Assay kit (Cayman, Ann Arbor, MI, USA). Retinas were mechanically lysed in lysis buffer (RayBiotech) containing a protease inhibitor cocktail and the antioxidant butyl hydroxytoluene (BHT). Lipid peroxidation was quantified sprectophotometrically at 540 nm (Thermo spectronic). MDA level was normalized per mg of protein present in each sample.
Protein nytrosilation was quantified in retinal lysates containing a protease inhibitor cocktail, measuring nitrotyroxine level with the 3-Nitrotyrosine ELISA kit (Abcam, Cambridge, UK) following the manufacturer’s instructions. The nitrotyroxine level was normalized per mg of protein present in each sample.
DNA oxidation was measured in genomic DNA isolated from retinal samples by DNAzol (Invitrogen). DNA was digested by incubation with DNAsa RQ1 (Promega, Madison, WI, USA) and 8-OHdG levels were quantified using the Oxidative DNA damage ELISA kit, following the manufacturer’s instructions. 8-OHdG levels were normalized per μg of DNA present in each sample.
For all oxidative markers, data were expressed as fold-change versus normal mice.