Immunofluorescence Analysis of Circadian Protein Dynamics in Cultured Mammalian Cells

The timing of both entry and permanence of core-clock proteins in the nucleus is critical to maintain the correct pace of the clock mechanism. Several such proteins, namely CRYPTOCHROMEs (CRY), PERIODs (PER), and BMAL1, were recently shown to contain nuclear transport signals that facilitate their “shuttling” between the nucleus and the cytoplasm. This type of dynamic intracellular movement not only regulates protein localization, but also often affects functions by determining interactive partners and protein turnover. Because most clock genes have been identified by genetic screening in Drosophila and by gene knockdown in mammals, it is important to develop cellular techniques to study the structure-function and regulation of the corresponding proteins. Here we present working protocols for immunofluorescence studies of clock proteins in mammalian cultured cells. This technique allows the visualization in the cell of one or multiple proteins at the same time.