Analysis of Histone Exchange during Chromatin Purification.

Abstract

Central to the study of chromosome biology are techniques that permit the purification of small chromatin sections for analysis of associated DNA and proteins, including histones. Chromatin purification protocols vary greatly in the extent of chemical cross-linking used to prevent protein dissociation/re-association during isolation. Particularly for genome-wide analyses, chromatin purification requires a balanced level of fixation that captures native protein-protein and protein/DNA interactions, yet leaving chromatin sections soluble and accessible to affinity reagents. We have applied a relative quantification methodology called I-DIRT (isotopic differentiation of interactions as random or targeted) for optimizing levels of chemical cross-linking for affinity purification of cognate chromatin sections. We show that fine-tuning of chemical cross-linking is necessary for isolation of chromatin sections when minimal histone/protein exchange is required.