title: Affinity purification of protein complexes from Drosophila embryos in cell cycle studies.
abstract: The ability to identify protein interactions is key to elucidating the molecular mechanisms of cellular processes, including mitosis and cell cycle regulation. Drosophila melanogaster, as a model system, provides powerful tools to study cell division using genetics, microscopy, and RNAi. Drosophila early embryos are highly enriched in mitotic protein complexes as their nuclei undergo 13 rounds of rapid, synchronous mitotic nuclear divisions in a syncytium during the first 2 h of development. Here, we describe simple methods for the affinity purification of protein complexes from transgenic fly embryos via protein A- and green fluorescent protein-tags fused to bait proteins of interest. This in vivo proteomics approach has allowed the identification of several known and novel mitotic protein interactions using mass spectrometry, and it expands the use of the Drosophila model in modern molecular biology.
authors.affiliation: Department of Genetics, University of Cambridge, Cambridge, UK.
authors.forename: Zoltan
authors.initials: Z
authors.lastname: Lipinszki
authors.forename: Peng
authors.initials: P
authors.lastname: Wang
authors.forename: Rhys
authors.initials: R
authors.lastname: Grant
authors.forename: Catherine
authors.initials: C
authors.lastname: Lindon
authors.forename: Nikola S
authors.initials: NS
authors.lastname: Dzhindzhev
authors.forename: Pier Paolo
authors.initials: PP
authors.lastname: D'Avino
authors.forename: Marcin R
authors.initials: MR
authors.lastname: Przewloka
authors.forename: David M
authors.initials: DM
authors.lastname: Glover
authors.forename: Vincent
authors.initials: V
authors.lastname: Archambault
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D002453:Cell Cycle; D002846:Chromatography, Affinity / Q000379:methods; D004330:Drosophila / Q000196:embryology* / Q000235:genetics / Q000378:metabolism; D029721:Drosophila Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D029721:Drosophila Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins