Immunoaffinity purification combined with 32P-postlabelling for the detection of O6-methylguanine in DNA from human tissues.
| dc.contributor.author | Cooper, Donald P | |
| dc.contributor.author | Griffin, Kathryn A | |
| dc.contributor.author | Povey, Andrew C | |
| dc.date.accessioned | 2010-08-04T09:54:14Z | |
| dc.date.available | 2010-08-04T09:54:14Z | |
| dc.date.issued | 1992-03 | |
| dc.identifier.citation | Immunoaffinity purification combined with 32P-postlabelling for the detection of O6-methylguanine in DNA from human tissues. 1992, 13 (3):469-75 Carcinogenesis | en |
| dc.identifier.issn | 0143-3334 | |
| dc.identifier.pmid | 1547539 | |
| dc.identifier.doi | 10.1093/carcin/13.3.469 | |
| dc.identifier.uri | http://hdl.handle.net/10541/109027 | |
| dc.description.abstract | Three 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. | |
| dc.language.iso | en | en |
| dc.subject.mesh | Chromatography, Thin Layer | |
| dc.subject.mesh | DNA | |
| dc.subject.mesh | Guanine | |
| dc.subject.mesh | Humans | |
| dc.subject.mesh | Phosphorus Radioisotopes | |
| dc.title | Immunoaffinity purification combined with 32P-postlabelling for the detection of O6-methylguanine in DNA from human tissues. | en |
| dc.type | Article | en |
| dc.contributor.department | Cancer Research Campaign Department of Carcinogenesis, Paterson Institute for Cancer Research, Manchester, UK. | en |
| dc.identifier.journal | Carcinogenesis | en |
| html.description.abstract | Three 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. |