Identification of Sequence-Specific DNA Binding Proteins

Abstract

Publisher Summary This chapter describes methods for the identification of nuclear proteins that bind specific DNA sequences within the cis -regulatory domains of genes. Protocols for the preparation of nuclear extract, quantitative electrophoretic mobility shift analysis, oligonucleotide affinity chromatography, and protein preparation for amino acid sequence analysis are presented, along with examples of the successful application of these methods to the cis -regulatory analysis of the Cyllla and Endo16 genes. It is expected that with the completion of the Strongylocentrotus purpuratus genome project and the availability of highly sensitive methods of protein sequencing by mass spectrometry, the biochemical approach described here for the identification of transcription factors will become even more efficient and adaptable to rapid high-throughput technologies. The studies of the sea urchin embryo have provided a paradigm for the molecular analysis of transcriptional regulatory systems that control development. Sea urchin embryos are extremely amenable to gene transfer by microinjection and also it readily provides the large quantities of raw material required for protein biochemistry. This makes it possible to functionally dissect the cis -regulatory DNA sequences that control transcription during development, and in parallel, to identify the trans -acting factors that interact with each regulatory sequence. This chapter presents a methodology for the latter, from the preparation of nuclear extracts through the identification by the electrophoretic mobility shift analysis of specific sites of protein-DNA interaction, to the purification and identification of the proteins that bind that site.