8 ohdg dna damage elisa kit

DNA damage was assessed by the content of 8-hydroxydeoxyguanosine (8-OHdG) as described recently by Changou et al.58 (link). Extracted gDNA (1 μg) was converted to single-stranded DNA by incubating the sample at 95 °C for 5 min and rapidly chilling on ice. DNA samples were digested to nucleosides by incubating the denatured DNA with nuclease P1 (5U) for 2 h at 37 °C in sodium acetate (20 mM) pH 5.2, followed with treatment of alkaline phosphatase (5U, New England Biolabs) for 1 h at 37 °C in Tris (100 mM), pH 7.5. The reaction mixture was centrifuged for 5 min at 6,000 g and the supernatant was used for the 8-OHdG DNA damage ELISA kit (Cell Biolabs) according to supplier’s protocol.

We evaluated the concentration of 8-hydroxydeoxyguanosine (8-OHdG) using an 8-OHdG DNA damage ELISA kit (Cell Biolabs, San Diego, CA, USA), according to manufacturer’s instructions. Briefly, isolated DNA was denatured at 95 °C for 5 min and immediately transferred to ice. A total of 10 units of nuclease P1 and 20 mM sodium acetate (pH 5.2) were added to total DNA, and digested DNA to nucleosides for 2 h at 37 °C. Next, alkaline phosphatase (Takara, Japan) added for 15 min at 37 °C, followed by incubation for 15 min at 50 °C in 100 mM Tris buffer (pH 7.5). The reaction mixtures were centrifuged for 5 min at 6000× g and the supernatants were extracted for assay analysis. Briefly, 50 µL of each sample is treated in wells and incubated for 10 min at room temperature. An amount of 50 µL of anti-8-OHdG antibody is treated in each well and incubated for 1 h at room temperature on orbital shaker. Each well is rinsed 3 times, 100 µL secondary antibody is added in each well and incubated for 1 h at room temperature. After incubation, 100 µl of substrate solution buffer is added in each well for 20 min at room temperature and then the reaction is stopped by adding stop solution. The absorbance of 8-OHdG was measured using a microplate reader at 450 nm (VERSA max™, Molecular Devices, San Jose, CA, USA).

The DNA was extracted from about 10⁸ yeast cells with a Yeast DNA Extraction Reagent Kit according to the manufacturer’s instructions (Thermo Scientific, Illkirch, France). The 8-oxo-guanine content was then examined by the 8-OHdG DNA Damage ELISA Kit according to the manufacturer’s instructions (Cell BioLabs, Paris, France).