8-hydroxy-2-deoxyguanosine (8-OHdG) is produced by the oxidative damage of DNA by reactive oxygen and nitrogen species and serves as an established marker of oxidative stress. Hydroxylation of guanosine occurs in response to both normal metabolic processes and a variety of environmental factors (i.e., anything that increases reactive oxygen and nitrogen species). Increased levels of 8-OHdG are associated with the aging process as well as with a number of pathological conditions including cancer, diabetes, and hypertension. Quantify 8-OHdG in urine, cell culture, plasma and other sample matrices using ICT’s DNA Damage ELISA Kit. This kit offers a quick incubation time, stable reagents, and a user-friendly protocol.
The ELISA utilizes an 8-hydroxy-2-deoxyguanosine-coated plate and an HRP-conjugated 8-OHdG specific detection antibody. This produces an assay with a useful range of 0.94 – 60 ng/mL, and a sensitivity of 0.59 ng/mL. Analyze your results using an absorbance plate reader.
1. Prepare the standard and samples in the Sample and Standard Diluent.
2. Add 50 µL of prepared standards and samples in triplicate to appropriate wells.
3. Add 50 µL of the diluted antibody preparation to the appropriate wells.
4. Cover plate with Plate Cover and incubate at room temperature (20-25°C) for 1 hour.
5. Dilute 10X Wash Buffer 1:10 with diH2O.
6. Wash plate 4 times with 1X Wash Buffer.
7. Add 100 µL TMB Substrate to each well.
8. Cover plate with Plate Cover and incubate at room temperature in the dark for 30 minutes to develop the signal.
9. Add 100 µL Stop Solution to each well.
10. Measure absorbance using a plate reader at 450 nm.
11. Plot the standard curve and calculate sample concentrations.
Urine samples were spiked with 8-OHdG, diluted, and analyzed using the 8-OHdG ELISA Kit. The y-intercept corresponds to the amount of 8-OHdG in unspiked urine. Error bars represent standard deviations obtained from multiple dilutions of each sample.
• Target: 8-hydroxy-2-deoxyguanosine (8-OHdG)
• Method of Analysis: Absorbance Plate Reader
• Types of Samples: Culture medium, urine, plasma/serum, cell lysates, tissue samples, saliva
• Storage: 8-OHdG at -20°C, all other kit components at 2-8°C
• Shipping: Ships overnight (domestic), International Priority Shipping
• 8-OHdG: BSA Coated 96-Well Plate, #270
• 2 Plate Covers, #271
• 8-OHdG, 100 µL at 3.06 µg/mL, #6646
• 8-OHdG HRP Conjugated Monoclonal Antibody, 75 µL, #6647
• Sample and Standard Diluent, 50 mL, #6648
• 8-OhdG Antibody Diluent, 13 mL, #6649
• 10X Wash Buffer Concentrate, 50 mL, #6650
• TMB Substrate, 13 mL, #6651
• Stop Solution, 13 mL, #6652
• Kit Manual