Immunoaffinity purification combined with 32P-postlabelling for the detection of O6-methylguanine in DNA from human tissues.
AffiliationCancer Research Campaign Department of Carcinogenesis, Paterson Institute for Cancer Research, Manchester, UK.
MetadataShow full item record
AbstractThree different activated supports were used to immobilize alpha-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/mg 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 alpha-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 O6-MedG levels of 0.39, 0.38 and 0.45 mumol/mol 2'-deoxyguanosine were found. The minimum detection limit of the combined method is expected to be approximately 1 O6-MedG in 10(8) normal 2'-deoxyguanosines (i.e. approximately 30 lesions per human cell) when 100 micrograms of DNA is used.
CitationImmunoaffinity purification combined with 32P-postlabelling for the detection of O6-methylguanine in DNA from human tissues. 1992, 13 (3):469-75 Carcinogenesis
- The development, validation and application of a 32P-postlabelling assay to quantify O6-methylguanine in human DNA.
- Authors: Povey AC, Cooper DP
- Issue date: 1995 Jul
- Optimization of 32P-postlabelling assays for the quantitation of O6-methyl and N7-methyldeoxyguanosine-3'-monophosphates in human DNA.
- Authors: Haque K, Cooper DP, Povey AC
- Issue date: 1994 Nov
- Relationships between the formation of O6-methyldeoxyguanosine by 1-p-carboxyl-3,3-dimethylphenyltriazene in DNA and O6-alkylguanine-DNA alkyltransferase in human peripheral leukocytes.
- Authors: Lee SM, O'Connor PJ, Thatcher N, Crowther D, Margison GP, Cooper DP
- Issue date: 1994 Aug 1
- Radioimmunoassay of O6-methyldeoxyguanosine in DNA of cells alkylated in vitro and in vivo.
- Authors: Wild CP, Smart G, Saffhill R, Boyle JM
- Issue date: 1983 Dec
- Quantitative determination of N7-methyldeoxyguanosine and O6-methyldeoxyguanosine in DNA by LC-UV-MS-MS.
- Authors: Yang Y, Nikolic D, Swanson SM, van Breemen RB
- Issue date: 2002 Oct 15