Co-immunoprecipitation: Protein–RNA and Protein–DNA Interaction

Abstract

Transcriptional and posttranscriptional regulators play a critical role in allowing a bacterium to adapt to the diverse environments and conditions it encounters. In order to characterize the role of these regulators the identification of their specific interaction partners is of utmost importance. Co-immunoprecipitation (IP) is based on antigen/antibody complex formation to purify a protein of interest from the rest of the samples together with its interaction partner. This method allows us to study direct interaction of a regulator with its specific binding partners like protein–RNA, protein–DNA, or protein–protein interactions. IP typically requires careful optimization and troubleshooting depending on the varying physicochemical characteristics of the protein of interest. In this chapter we present a starting point and the basic guidelines to obtain the best possible results from an IP experiment with subsequent use of new-generation sequencing techniques to detect mRNA or ncRNA targets (RIPseq) and protein–DNA interactions (ChIPseq).