Isolation and Immunodetection of Enzymatic DNA-Protein Crosslinks by RADAR Assay.

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.