Home > Protocols > Detection of 8-OHdG in tissue samples Rev.090309
Detection of 8-hydroxy-2'-deoxyguanosine in tissue/cultured cells.
This is an application example to detect 8-OHdG in tissue samples or cultured cells. Experimental conditions should be settled by each user.
1) Extract DNA from cells
Dextract DNA from the homogenates using a DNA extraction kit. Please follow the manufacture's manual.

Followings are DNA extraction kits suitable for 8-OHdG detection, and are available from WAKO Chemicals USA Inc. and from WAKO Chemicals GmbH (Germany).
1) DNA Extractor® WB Kit (Sodium Iodide Method): cat#291-50502
2) DNA Extractor® TIS Kit (Sodium Iodide Method): cat#296-67701
3) 8-OHdG Assay Preparation Reagent Set: cat#292-67801
2) Calculation of DNA concentration and purity
a) DNA Solution is diluted to 20-50 µg/mL with TE buffer.
b) Measure absorbance at wavelength 230nm, 260nm, 280nm and 320nm.
c) Calculate DNA Concentration. (1.0 of Absorbance at 260nm is equal to 50 µg/mL DNA)
DNA concentration (µg/mL) = (Absorbanve at 260nm) * (50 µg/mL) * dilution rate
d) Check the purity of DNA sample.
The ratio of (Absorbance at 260nm) : (Absorbance at 280nm) is typically between 1.8 to 1.85.
The purity of isolated DNA is assessed by the ratio of (Absorbance at 260nm) : (Absorbance at 230nm), and should be between 2.2 to 2.25.
3) Enzymatic digestion of DNA
a) Dissolve 200 µg of extracted DNA in 135 µL of water.
b) Add 15 micro L of 200mM sodium acetate and 6 units of nuclease P1 (15 µL, 1 mg/mL) to the DNA Solution, and then incubated for 30 min to 1hr at 37 °C after Argon substitution.
c) Add 1M Tris-HCl buffer (15 µL, pH 7.4) and 2 unit of alkaline phosphatase (7 µL, 200 units/0.7mL), and incubated for 30 min to 1hr at 37 °C after Argon substitution.
d) To remove enzymes and other macromolecules, the hydrolysate are filtered through Millipore Microcon YM-10(catalog#42407) at 14000rpm for 10min.
e) Apply 50 µL of DNA digest to the wells of ELISA kit. It is recommended that analysis procedure mentioned here shoukd be completed in one day.
  NOTE:
1) Enzyme-treated samples should be applied in the same day.
2) TE buffer: 1mM EDTA / 10mM Tris-HCl, pH8.0
3) Water used should be treated by Chelex (Bio Rad) to remove trace metal ions.
References
1) ML Hamilton, Z Guo, CD Fuller, H Van Remmen, WF Ward, SN Austad, DA Troyer, I Thompson, A Richardson:
A reliable assessment of 8-oxo-2-deoxyguanosine levels in nuclear and mitochondrial DNA using the sodium iodide method to isolate DNA.
Nucleic Acids Res. Vol.29(10), p2117-2126, 2001
2) S Toyokuni, et. al.:
Quantitative Immunohistochemical determination of 8-hydroxy-2'-deoxyguanosine by a monoclonal antibody N45.1: Its application to ferric nitrilotriacetate-induced renal carcinogenesis model.
Laboratory Investigation, Vol. 76, No.3, p365-374, 1997
3) M Kakimoto, T Inoguchi, T Sonta, HY Yu, M Imamura, T Etoh, T Hashimoto and H Nawata:
Accumulation of 8-hydroxy-2'-deoxyguanosine and mitochondrial DNA deletion in kidney of diabetic rats.
Diabetes, Vol. 51, p1588-1595, 2002
4) D Nakae, Y Mizumoto, E Kobayashi, O Noguchi and Y Konishi:
Improved genomic / nuclear DNA extraction for 8-hydroxydeoxyguanosine analysis of small amounts of rat liver tissue.
Cancer Lett., Vol. 97, p233-239, 1995
Highly Sensitive 8-OHdG Check ELISA Technical Information
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