title: Affinity filtration coupled with capillary-based affinity purification for the isolation of protein complexes.
abstract: The isolation of complex macromolecular assemblies at the concentrations required for structural analysis represents a major experimental challenge. Here we present a method that combines the genetic power of site-specific recombination in order to selectively "tag" one or more components of a protein complex with affinity-based rapid filtration and a final step of capillary-based enrichment. This modified form of tandem affinity purification produces highly purified protein complexes at high concentrations in a highly efficient manner. The application of the method is demonstrated for the yeast Arp2/3 heptameric protein complex involved in mediating reorganization of the actin cytoskeleton.
authors.affiliation: Krebs Institute, Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield, UK.
authors.forename: M S
authors.initials: MS
authors.lastname: Qureshi
authors.forename: Q I
authors.initials: QI
authors.lastname: Sheikh
authors.forename: R
authors.initials: R
authors.lastname: Hill
authors.forename: P E
authors.initials: PE
authors.lastname: Brown
authors.forename: M J
authors.initials: MJ
authors.lastname: Dickman
authors.forename: S B
authors.initials: SB
authors.lastname: Tzokov
authors.forename: D W
authors.initials: DW
authors.lastname: Rice
authors.forename: D T
authors.initials: DT
authors.lastname: Gjerde
authors.forename: D P
authors.initials: DP
authors.lastname: Hornby
mesh_terms: D051376:Actin-Related Protein 2-3 Complex / Q000302:isolation & purification; D002846:Chromatography, Affinity / Q000379:methods*; D005374:Filtration / Q000379:methods*; D011506:Proteins / Q000302:isolation & purification*; D029701:Saccharomyces cerevisiae Proteins / Q000302:isolation & purification
chemical_list: D051376:Actin-Related Protein 2-3 Complex; D011506:Proteins; D029701:Saccharomyces cerevisiae Proteins