Oxiselect hne his adduct elisa kit

4-HNE Protein adducts and MDA levels were measured in mice to determine whether toxic lipid peroxidation aldehydes would be produced in response to hyperoxia-induced oxidative stress. C57BL/6J mice (N = 6) were exposed to normoxia, as well as hyperoxia for 24, 48, and 72 hours. Lungs were isolated and perfused with cold 0.9% NaCl with 0.1% glucose, 30 mM HEPES, 6mM KCl, 0.1 mg/ml streptomycin sulfate, 0.07 mg/ml penicillin G, 0.07 mg/ml EGTA, and 20 mM Tris-HCl (pH 7.4), as previously described [9 (link)]. Homogenates were then centrifuged at 3,000g for 10 min at 4°C and 200 μL of the supernatant was removed. 4-HNE-Protein adduct levels were measured by an OxiSelect HNE-His Adduct ELISA kit, according to manufacturer's instructions (Cell Biolabs, Inc., San Diego, CA). On a 96-well plate, protein samples and standards (10 μg/ml) were absorbed for 2 hrs at 37°C. Protein was probed with anti-HNE Histidine (His) antibody, followed by horseradish peroxidase secondary antibody. Protein levels were compared with a standard curve produced from HNE-BSA standards. MDA levels were measured in mice lung homogenates with a Lipid Peroxidation (MDA) Assay Kit (Colorimetric/Fluorometric) ab118970 (abcam, Cambridge, MA), after washing with cold PBS. MDA levels were also compared with a MDA standard curve, prepared from MDA Standard.