title: Purification of ribonucleoproteins using peptide-elutable antibodies and other affinity techniques.
abstract: 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 cerevisiae. 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 degrees 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.
authors.affiliation: Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas, USA.
authors.forename: Scott W
authors.initials: SW
authors.lastname: Stevens
mesh_terms: D000906:Antibodies / Q000737:chemistry*; D002846:Chromatography, Affinity / Q000379:methods*; D016681:Genome, Fungal; D010455:Peptides / Q000737:chemistry*; D010957:Plasmids / Q000235:genetics; D012261:Ribonucleoproteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D012441:Saccharomyces cerevisiae / Q000235:genetics
chemical_list: D000906:Antibodies; D010455:Peptides; D012261:Ribonucleoproteins