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Preparation of Nuclear and Cytoplasmic Extracts from Mammalian Cells

Extracts prepared from the isolated nuclei of cultured cells are functional in accurate in vitro transcription and mRNA processing. Thus, such extracts can be used directly for functional studies and as the starting material for purification of the proteins involved in these processes. In this unit, reproducible extracts from different preparations of nuclei are generated. The conditions described are optimized to produce transcriptionally active extracts from HeLa cells. A support protocol describes how to optimize the basic procedure to increase the yield of specific proteins or activities from different cell types, and a second support protocol describes the preparation of the cytoplasmic (S‐100) fraction.