A Method to Separate Nuclear, Cytosolic, and Membrane-Associated Signaling Molecules in Cultured Cells

Direct comparison of protein distribution between the nucleus, the cytoplasm, and the plasma membrane is important for understanding cellular processes and, in particular, signal transduction, where cascades generated at the cell surface regulate functions in other cellular compartments, such as the nucleus. Yet, many commonly used methods fail to effectively separate the plasma membrane and the cytoskeleton from the nucleus, and the cytosol from the nucleosol. This problem has led to confounding results in the study of signaling pathways due to incorrect assignment of cellular localization to signaling molecules and presents challenges in the biochemical study of soluble proteins that shuttle between the cytoplasm and the nucleus. We present a simple method, based on partial membrane permeabilization with detergent followed by density gradient centrifugation, that provides rapid separation of cytosolic, nucleosolic, nuclear insoluble, and membrane components in various mammalian cell lines.