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Summary: Cells possess multiple DNA repair pathways to tackle a variety of DNA lesions. Often, DNA repair proteins function as large protein complexes. Here, we describe a protocol to purify DNA repair protein complexes from nuclei of mammalian cells. The method permits purification of protein complexes containing stable as well as transiently associated proteins, which subsequently can be identified by mass-spectrometry analysis. This protocol can be applied to uncover the functions and mechanism of DNA repair pathways.For complete information on the use and execution of this protocol, please refer to Socha et al. (2020).