Abstract
This chapter provides an overview of the analysis of messenger ribonucleoprotein (mRNP) complexes assembled in vitro. Analysis of sequence-specific interactions between Ribonucleic acid (RNA) and proteins is essential for defining the biochemistry of the posttranscriptional steps in gene expression and in understanding a broad range of corresponding genetic controls. Sequential steps in RNA synthesis and expression in which RNA–protein interactions are central include: (1) transcript splicing, (2) 3′ processing, nuclear-to-cytoplasmic transport, subcytoplasmic localization, (3) translational modulation, and (4) determination of messenger RNAs (mRNA) stability. The gel retardation or electromobility shift assay is the simplest and perhaps the most widely used method for investigating protein-nucleic acid interactions in vitro. This chapter discusses in detail about the complex formation including preparation of cytoplasmic S100 extracts, and preparation of RNA probe. Detection of RNP complexes is also elaborated. The chapter discusses about analysis of RNP complexes identified by electrophoretic mobility shift analysis. Direct analysis of protein constituents of ribonucleoprotein (RNP) complexes by transfer to membranes is elaborated in depth. The chapter also explains analysis of resolved complexes excised from the gel.
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