Abstract
This chapter discusses the basic preparations of reagents and their application to the analysis of transcription factor binding to nucleosomes. It provides protocols for the preparation of nucleosomes and histones, nucleosome reconstitution, and the analysis of transcription factor binding. The chapter also provides protocols that are applicable to any nucleosome-length DNA fragment that can be easily performed by any biochemist or molecular biologist with minimal protein experience. The only limitation to performing these experiments is the availability of sufficient quantities of the transcription factors of interest. The direct analysis of transcription factor binding to nucleosomes has revealed many general properties of factor-nucleosome interactions. Different factors are inhibited in binding to different degrees by the occupancy of their binding sites in nucleosomes. For example, the glucocorticoid receptor exhibits very efficient nucleosome binding abilities, whereas similar analyses indicate that the binding of NF1 and the human heat-shock factor are severely inhibited by nucleosomes. Other factors demonstrate intermediate levels of affinity for nucleosomal DNA.
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