title: Native Elution of Yeast Protein Complexes Obtained by Affinity Capture.
abstract: This protocol describes two options for the native (nondenaturing) elution of protein complexes obtained by affinity capture. The first approach involves the elution of complexes purified through a tag that includes a human rhinovirus 3C protease (PreScission protease) cleavage site sequence between the protein of interest and the tag. Incubation with the protease cleaves immobilized complexes from the affinity medium. The second approach involves the release of protein A-tagged protein complexes using a competitive elution reagent called PEGylOx. The degree of purity of the native assemblies eluted is sample dependent and strongly influenced by the affinity capture. It should be noted that the efficiency of native elution is commonly lower than that of elution by a denaturing agent (e.g., SDS) and the release of the complex will be limited by the activity of the protease or the inhibition constant (Ki) of the competitive release agent. However, an advantage of native release is that some nonspecifically bound materials tend to stay adsorbed to the affinity medium, providing an eluted fraction of higher purity. Finally, keep in mind that the presence of the protease or elution peptide could potentially affect downstream applications; thus, their removal should be considered.
authors.affiliation: Laboratory of Cellular and Structural Biology, The Rockefeller University, New York, New York 10065.
authors.forename: John
authors.initials: J
authors.lastname: LaCava
authors.affiliation: Laboratory of Cellular and Structural Biology, The Rockefeller University, New York, New York 10065.
authors.forename: Javier
authors.initials: J
authors.lastname: Fernandez-Martinez
authors.affiliation: Laboratory of Cellular and Structural Biology, The Rockefeller University, New York, New York 10065.
authors.forename: Michael P
authors.initials: MP
authors.lastname: Rout
mesh_terms: D000345:Affinity Labels / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods*; D046911:Macromolecular Substances / Q000302:isolation & purification*; D009097:Multienzyme Complexes / Q000302:isolation & purification*; D012441:Saccharomyces cerevisiae / Q000737:chemistry*; D029701:Saccharomyces cerevisiae Proteins / Q000302:isolation & purification*
chemical_list: D000345:Affinity Labels; D046911:Macromolecular Substances; D009097:Multienzyme Complexes; D029701:Saccharomyces cerevisiae Proteins