Protocol for nucleo-cytoplasmic fractionation of mammalian cells to study protein translocation via immunoblotting

Studying protein translocation is essential for understanding protein function and regulatory mechanisms, including the activity of Transcription Factors. Here, we present a protocol for isolating high-quality cytoplasmic and nuclear protein fractions from mammalian cells. We describe steps for separating cytoplasmic proteins, washing nuclei, and extracting nuclear proteins. We then detail procedures for quantifying proteins and analyzing their distribution by immunoblotting within a single immunoblot. This protocol is applicable to both suspension and adherent cell types. For complete details on the use and execution of this protocol, please refer to Sharma et al.^1,2,3.