Analysis of Chromatin Binding of Ectopically Expressed Proteins in Early Xenopus Embryos

Abstract

Xenopus embryos have long been used to show phenotypic effects following overexpression of proteins of interest such as transcription factors. Posttranslational modification of these proteins can dramatically alter the extent of the observed phenotype by inhibiting or enhancing protein activity. To determine the mechanisms controlling transcription factor activity, it is useful to compare relative levels of chromatin-bound protein, as this can reveal altered chromatin association in addition to changes in overall protein accumulation seen in the cytoplasm. Assaying protein binding to the bulk DNA described here compliments alternative assays such as electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) that measure site-specific DNA binding. This protocol describes a method to prepare and analyze chromatin and cytoplasmic extracts from embryos overexpressing proteins of interest, and it uses a robust fractionation procedure that results in clear separation of cytoplasmic tubulin from histone-H3 enriched chromatin. This assay for relative chromatin-bound protein is most suitable for comparing modified forms of a single protein (e.g., to investigate the effects of point mutations on chromatin association). Optimization is required for the specific protein of interest but guide ranges are provided.