Bio-Protocol (Dec 2020)
Native Co-immunoprecipitation Assay to Identify Interacting Partners of Chromatin-associated Proteins in Mammalian Cells
Abstract
Protein-protein interactions play key roles in nuclear processes including transcription, replication, DNA damage repair, and recombination. Co-immunoprecipitation (Co-IP) followed by western blot or mass spectrometry is an invaluable approach to identify protein-protein interactions. One of the challenges in the Co-IP of a protein localized to nucleus is the extraction of nuclear proteins from sub-nuclear fractions without losing physiologically relevant protein interactions. Here we describe a protocol for native Co-IP, which was originally used to successfully identify previously known as well novel topoisomerase 1 (TOP1) interacting proteins. In this protocol, we first extracted nuclear proteins by sequentially increasing detergent and salt concentrations, the extracted fractions were then diluted, pooled, and used for Co-IP. This protocol can be used to identify protein-interactome of other chromatin-associated proteins in a variety of mammalian cells.