Isolation and Immunodetection of Enzymatic DNA-Protein Crosslinks by RADAR Assay.
Isolation and Immunodetection of Enzymatic DNA-Protein Crosslinks by RADAR Assay.
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通过 RADAR 测定法分离和免疫检测酶促 DNA-蛋白质交联。
DOI：
10.1007/978-1-0716-3373-1_8
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发表时间：
2023
期刊：
影响因子：
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通讯作者：
Ghosal,Gargi
中科院分区：
文献类型：
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作者：
Perry,Megan;Ghosal,Gargi
DNA-蛋白质交联 (DPC) 是 DNA 代谢过程的空间障碍，DPC 的去除和修复是一个快速发展的研究领域。破译这一修复途径的一个关键部分是开发检测和量化细胞中特定类型 DPC 的技术。在这里，我们描述了一种直接检测哺乳动物细胞中酶促 DPC 的方案——RADAR 测定。该方法包括从细胞中分离基因组 DNA 和 DPC，并使用真空槽印迹歧管将它们结合到硝酸纤维素膜上。使用针对目标蛋白质产生的抗体来检测 DPC，并通过标准化为 DNA 上样对照来进行定量。 RADAR 测定可以检测特定类型的 DPC 并对各种药物的 DNA-蛋白质交联活性进行灵敏分析，可适应不同的细胞类型和条件，并且几乎不需要专门的设备。
DNA–protein crosslinks (DPCs) are steric hindrances to DNA metabolic processes and the removal and repair of DPCs is a rapidly evolving area of research. A critical component of deciphering this repair pathway is developing techniques that detect and quantify specific types of DPCs in cells. Here we describe a protocol for direct detection of enzymatic DPCs from mammalian cells—the RADAR assay. The method involves isolating genomic DNA and DPCs from cells and binding them to nitrocellulose membrane with a vacuum slot blot manifold. DPCs are detected using antibodies raised against the protein of interest and quantified by normalizing to a DNA loading control. The RADAR assay allows for the detection of specific types of DPCs and the sensitive analysis of the DNA–protein crosslinking activity of various drugs, is adaptable across different cell types and conditions, and requires little specialized equipment.