11] Methods for isolation and characterization of nonhistone chromosomal proteins

Abstract

Publisher Summary This chapter provides methods for the isolation and characterization of nonhistone chromosomal (NHC) proteins that are defined as proteins, other than the histones, which isolate in association with nuclear DNA in the purification of chromatin. The methods for the isolation and characterization of NHC proteins are developed using chromatin. The solubilized chromatin is the starting material for the isolation of NHC proteins. It is important to lyse the nuclei in buffers with low concentrations of salts. Lysis in 0.14 N NaC1 can apparently lead to adventitious adsorption of additional nuclear and cytoplasmic protein to the chromatin. The purification of chromatin by centrifugation through 1.7 M sucrose is important in eliminating loosely bound protein. That chromatin prepared by this method is equivalent to the complex as it exists in vivo is indicated by the fact that its properties as a template for RNA polymerase are the same, to the extent that this can be assessed with current technology. Several methods have been developed for the fractionation of chromatin into its three main constituent components—DNA, histones, and NHC proteins. The sodium dodecyl sulfate (SDS) method for the preparation of NHC proteins coupled with the analysis by SDS polyacrylamide disc gel electrophoresis gives very reproducible results in terms of both the quantitative and qualitative features of gel band pattern.