用于检测哺乳动物细胞线粒体dna -蛋白交联的酶联免疫吸附试验。

DNA Pub Date : 2022-12-01 DOI:10.3390/dna2040019
Wenyan Xu, Linlin Zhao
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引用次数: 4

摘要

DNA-蛋白质交联(DPCs)是一种细胞毒性的DNA损伤,蛋白质与DNA共价结合。尽管人们对细胞核中DPCs的形成、修复和生物学后果了解甚多,但对线粒体DPCs知之甚少。这部分是由于缺乏可靠和特定的方法来测量线粒体DPCs。在此,我们报道了一种基于酶联免疫吸附试验(ELISA)的方法,用于检测培养的人细胞中DNA和线粒体转录因子A (TFAM)之间形成的线粒体DPCs。为了优化纯化和检测流程,我们利用重组人TFAM和含有碱性(AP)病变的DNA底物,通过希夫碱化学制备了模型TFAM- dpcs。我们优化了用硅胶基柱分离TFAM-DPCs的方法,以获得高回收率的DPCs。我们评估了微孔板、dna包被液和HRP底物对TFAM-DPCs的特异性和敏感性。此外,我们优化了mtDNA的分离程序,以消除几乎所有的核DNA污染物。为了证明这一概念,我们检测了不同生物学条件下HEK293细胞mtDNA中不同水平的TFAM-DPCs。该方法基于商业上可用的材料,并且可以修改以检测线粒体中其他类型的DPCs。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

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An Enzyme-Linked Immunosorbent Assay for the Detection of Mitochondrial DNA-Protein Cross-Links from Mammalian Cells.

DNA-Protein cross-links (DPCs) are cytotoxic DNA lesions with a protein covalently bound to the DNA. Although much has been learned about the formation, repair, and biological consequences of DPCs in the nucleus, little is known regarding mitochondrial DPCs. This is due in part to the lack of robust and specific methods to measure mitochondrial DPCs. Herein, we reported an enzyme-linked immunosorbent assay (ELISA)-based method for detecting mitochondrial DPCs formed between DNA and mitochondrial transcription factor A (TFAM) in cultured human cells. To optimize the purification and detection workflow, we prepared model TFAM-DPCs via Schiff base chemistry using recombinant human TFAM and a DNA substrate containing an abasic (AP) lesion. We optimized the isolation of TFAM-DPCs using commercial silica gel-based columns to achieve a high recovery yield for DPCs. We evaluated the microplate, DNA-coating solution, and HRP substrate for specific and sensitive detection of TFAM-DPCs. Additionally, we optimized the mtDNA isolation procedure to eliminate almost all nuclear DNA contaminants. For proof of concept, we detected the different levels of TFAM-DPCs in mtDNA from HEK293 cells under different biological conditions. The method is based on commercially available materials and can be amended to detect other types of DPCs in mitochondria.

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DNA
DNA
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