Chapter 4 Methods for Isolation of Nuclei from Cultured Mammalian Cells; Conditions for Preferential Retention of Selected Histones

Abstract

This chapter describes the methods for isolation of nuclei from cultured mammalian cells; conditions for preferential retention of selected histones. Histones serve as useful standards to assay for leakage of nuclear components as a function of the composition of the media used for cell lysis and isolation of nuclei. The chapter discusses the methods to study two kinds of histones: (1) lysine-rich and slightly lysine-rich histones, and (2) arginine-rich histones. None of the methods used for cell lysis prior to isolation of nuclei and chromatin conserves all the histones. The arginine-rich histones are poorly conserved by methods that use chelating agents, such as ethylenediaminetetraacetic acid (EDTA) or citrate, in the absence of appropriate concentrations of divalent cations. A neutral pH method that minimizes leakage of arginine-rich histones through the addition of 10 mM Mg 2+ and Ca 2+ has been developed.