@inbook{26acd48ab87f4c30afd6087d4444519b,
title = "Isolation and Immunodetection of Enzymatic DNA–Protein Crosslinks by RADAR Assay",
abstract = "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.",
keywords = "DNA repair, DNA-protein crosslink, DPC repair, RADAR assay, Slot blot, TOP1-cc, TOP2-cc",
author = "Megan Perry and Gargi Ghosal",
note = "Funding Information: This work was supported by NIGMS COBRE (P20 GM121316; G.G. as project leader), NCI K22 Career Development Award (K22CA188181) to G.G., R01 grants GM141232-01A1 and CA263504-01A1 to G.G. Publisher Copyright: {\textcopyright} 2023, The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.",
year = "2023",
doi = "10.1007/978-1-0716-3373-1_8",
language = "English (US)",
series = "Methods in Molecular Biology",
publisher = "Humana Press Inc.",
pages = "135--148",
booktitle = "Methods in Molecular Biology",
}