کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
5511083 1539377 2017 17 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
An improved method for the detection of nucleotide excision repair factors at local UV DNA damage sites
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی زیست شیمی
پیش نمایش صفحه اول مقاله
An improved method for the detection of nucleotide excision repair factors at local UV DNA damage sites
چکیده انگلیسی
Among different DNA repair processes that cells use to face with DNA damage, nucleotide excision repair (NER) is particularly important for the removal of a high variety of lesions, including those generated by some antitumor drugs. A number of factors participating in NER, such as the TFIIH complex and the endonuclease XPG are also involved in basal processes, e.g. transcription. For this reason, localization of these factors at DNA damage sites may be difficult. Here we have applied a mild digestion of chromatin with DNase I to improve the in situ extraction necessary to detect chromatin-bound proteins by immunofluorescence. We have compared this method with different extraction protocols and investigated its application on different cell types, and with different antibodies. Our results show that a short DNase I treatment before the immunoreaction, enhances the fluorescence signal of NER proteins, such as XPG, DDB2 and XPC. In addition, our findings indicate that the antibody choice is a critical factor for accurate localization of DNA repair proteins at DNA damage sites. In conclusion, a mild DNA digestion with DNase I improves the immunofluorescence detection of the recruitment of NER factors at local DNA damage sites by enhancing accessibility to the antibodies, independently of the cell type.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: DNA Repair - Volume 51, March 2017, Pages 79-84
نویسندگان
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