Purification of Ribonucleoproteins Using Peptide-Elutable Antibodies and Other Affinity Techniques

Download Book (13,976 KB) As a courtesy to our readers the eBook is provided DRM-free. However, please note that Springer uses effective methods and state-of-the art technology to detect, stop, and prosecute illegal sharing to safeguard our authors’ interests.
Download Protocol (507 KB)

Summary

Recently developed affinity purification methods have revolutionized our understanding of the higher-ordered structures of multisubunit, often low-abundance macromolecular complexes, including ribonucleoproteins (RNPs). Often, purification by classical, non-affinity-based techniques subjects salt-labile complexes to an ionic strength incompatible with the integrity of the RNP, leading to a misrepresentation of the true higher-ordered structure of these complexes. A family of plasmids has been generated that can be used to introduce a number of different epitope tags, including peptide-elutable affinity tags, into the genome of the yeast Saccharomyces cerevi-siae. Alternatively, these plasmids may be used for plasmid-borne expression of epitope-tagged proteins in either yeast or Escherichia coli. The gentle elution of the complex from the antibody affinity matrix can be performed at 4 °C and is compatible with a range of salt and pH conditions. RNPs purified by this method are active and suitable for downstream analyses such as RNA sequencing, structural analysis, or mass spectrometry peptide identification.