Analysis of chromatin binding of ectopically expressed proteins in early Xenopus embryos

Xenopus embryos have long been used to demonstrate phenotypic effects following overexpression of proteins of interest such as transcription factors, while post-translational modification of these proteins can dramatically alter the extent of the observed phenotype by inhibiting or enhancing protein activity. In order 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 EMSA and ChIP that measure sitespecific DNA binding. This protocol describes a method to prepare and analyse chromatin and cytoplasmic extracts from embryos over-expressing the proteins of interest, and 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, for example to investigate the effects of point mutations on chromatin association. Optimisation is required for the specific protein of interest but guide ranges are provided.L.H. was funded by a Medical Research Council Studentship. Research in AP’s laboratory is supported by Medical Research Council grants MR/K018329/1, MR/L021129/1, and core support from the Wellcome Trust and MRC to the Wellcome Trust – Medical Research Council Cambridge Stem Cell Institute. LH is supported by a Peterhouse Fellowship