title: A generic protocol for the purification and characterization of water-soluble complexes of affinity-tagged proteins and lipids.
abstract: Interactions between lipids and proteins in the aqueous phases of cells contribute to many aspects of cell physiology. Here we describe a detailed protocol to systematically characterize in vivo-assembled complexes of soluble proteins and lipids. Saccharomyces cerevisiae strains expressing physiological amounts of a protein of interest fused to the tandem-affinity purification (TAP) tag are first lysed in the absence of detergent to capture intact protein-lipid complexes. The affinity-purified complexes (typically 30-50 kDa) are subjected to analytical size-exclusion chromatography (SEC) to remove contaminating lipids that elute at the void volume (>600 kDa), in order to achieve sufficient signal-to-background lipid ratios. Proteins in the SEC fractions are then analyzed by denaturing gel electrophoresis. Lipidomics techniques such as high-performance thin-layer chromatography or gas or liquid chromatography-mass spectrometry can then be applied to measure the elution profiles of lipids and to pinpoint the true interactors co-eluting with the TAP fusions. The procedure (starting from cell lysis) requires 2 d, and it can easily be adapted to other organisms.
authors.affiliation: Structural and Computational Biology Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany.
authors.forename: Kenji
authors.initials: K
authors.lastname: Maeda
authors.affiliation: Structural and Computational Biology Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany.
authors.forename: Mattia
authors.initials: M
authors.lastname: Poletto
authors.affiliation: Structural and Computational Biology Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany.
authors.forename: Antonella
authors.initials: A
authors.lastname: Chiapparino
authors.affiliation: Structural and Computational Biology Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany.
authors.forename: Anne-Claude
authors.initials: AC
authors.lastname: Gavin
mesh_terms: D002623:Chemistry Techniques, Analytical / Q000379:methods*; D002850:Chromatography, Gel / Q000379:methods; D002855:Chromatography, Thin Layer; D058645:Denaturing Gradient Gel Electrophoresis / Q000379:methods; D008401:Gas Chromatography-Mass Spectrometry; D007553:Isotope Labeling / Q000379:methods; D008055:Lipids / Q000737:chemistry* / Q000302:isolation & purification; D046911:Macromolecular Substances / Q000302:isolation & purification*; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry*; D014867:Water
chemical_list: D008055:Lipids; D046911:Macromolecular Substances; D011506:Proteins; D014867:Water