Immunoaffinity purification combined with 32P-postlabelling for the detection of O6-methylguanine in DNA from human tissues
- 1 March 1992
- journal article
- research article
- Published by Oxford University Press (OUP) in Carcinogenesis: Integrative Cancer Research
- Vol. 13 (3) , 469-475
- https://doi.org/10.1093/carcin/13.3.469
Abstract
Three different activated supports were used to immobilize α-O6-methyldeoxyguanosine (O6-MedG) monoclonal antibody. Affinity gels based on CNBr Sepharose, Affigel Hz and periodate-activated Sepharose (PIAS) were able to bind 1.4, 1.5 and 6.2 nmol [3H]O6-MedG/ immobilized IgG respectively, and recovery of bound material exceeded 95% in all cases. Significant non-specific binding of normal nucleosides occurred only when using the CNBr gel. PIAS α-O6-MedG affinity gel was able to purify O6-MedG-3′-monophosphate from digested synthetic oligonucleotides and in vitro-methylated calf thymus DNA to allow subsequent detection by 32P-postlabelling and two-dimensional TLC. The combined method was applied to three human samples and O6P-postlabelling and two-dimensional TLC. The combined method was applied to three human sample and O6-MedG levels of 0.39, 0.38 and 0.45 μmol/mol 2′-deoxyguanosine were found. The minimum detection limit of the combined method is expected to be ∼1 O6-MedG in 108 normal 2′-deoxyguanosines (i.e. ∼30 lesions per human cell) when 100 μg of DNA is used.Keywords
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