Chapter 18 In Vitro Nuclear Protein Import Using Permeabilized Mammalian Cells

Publisher Summary This chapter discusses the in vitro nuclear protein import using permeabilized mammalian cells. The nuclear import system is simple and efficient and can be tailored to the needs of the individual laboratory. A wide range of different cell types can be used as the source of permeabilized cells or cytosol. A fluorescent reporter protein is utilized to measure nuclear import because it is possible to visualize directly its nuclear accumulation and determine the range of nuclear accumulation given by a population of nuclei. An allophycocyanin is used for this assay because of its large size, its naturally high fluorescence output, and its resistance to photobleaching. Another widely used protein for the analysis of nuclear import is Xenopu s nucleoplasmin—a nuclear protein that is easily purified to homogeneity in sufficient quantities. The rhodamine-labeled nucleoplasmin exhibits high nonspecific binding to the permeabilized cells and is imported weakly. A nonfluorescent reporter protein is also used to measure import if an antibody to the protein is available to measure its nuclear accumulation by immunofluorescence microscopy, although the protein must not be present in the cells used for the assay and the analysis is lengthened by the requisite immunofluorescence labeling.