Chapter 2 Nonaqueous Isolation of Nuclei from Cultured Cells

Abstract

Publisher Summary This chapter describes the nonaqueous isolation of nuclei from cultured cells. The nonaqueous method of cell fractionation helps for bulk separation of cell components while avoiding the extraction or redistribution of water-soluble material encountered in conventional aqueous fractionation. Nonaqueous isolation of nuclei can be used to determine the species and concentrations of soluble nuclear components, including ions and soluble nuclear proteins. Nonaqueous nuclear isolation consists of lyophilization of tissues or dispersed cells, homogenization of the dry powder in a nonaqueous liquid, and then sedimentation of nuclei, again in a nonaqueous liquid. Kirsch and others have made three important refinements of the nonaqueous method: (1) by substituting dichlorodifluoromethane for isopentane in a histochemical freezing method, (2) by chilling frozen samples during the drying, and (3) by using glycerol as the nonaqueous homogenization medium, replacing the petroleum ether, benzene, and cyclohexane of older methods. Several simplifications make the procedure easy without compromising the quality of isolated nuclei. The methods show that DNA polymerase-α, can be quantitatively recovered from nuclei of cultured mouse and human cells.