13] Methods for isolation and characterization of acidic chromatin proteins

Abstract

Publisher Summary The increasing awareness about the role of acidic chromatin proteins as specific gene regulators has been obscured by difficulties inherent in their isolation and characterization. This chapter describes a rapid and quantitative isolation of chromatin acidic proteins by three simple steps—namely, acid dehistonization of chromatin, hydrolysis of DNA by a short incubation with bovine pancreatic DNase I, and removal of soluble DNA fragments by acid that precipitates the protein. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the isolated proteins reveals reproducible heterogeneous banding patterns, which appear to be tissue specific. The DNase procedure described for isolating the acidic proteins of chromatin is advantageous in that it is quick, quantitative, and reproducible. The necessity of removing DNA from chromatin preparations for effective resolution of the acidic proteins on SDS polyacrylamide gels is demonstrated in the chapter. Electrophoresis of whole chromatin results in streaking and faint bands, in contrast to the distinct bands of a preparation of dehistonized chromatin that, prior to gel electrophoresis, is treated with DNase.