title: Purification of Drosophila protein complexes for mass spectrometry.
abstract: Drosophila melanogaster is one of the best characterized model systems for genetic analysis. Protein biochemical methods have lagged behind for quite some time but meanwhile have reached a state where protein interaction networks can be elucidated at a similar speed and accuracy as genetic interactions. Therefore, Drosophila now offers the advantages of both genetic and biochemical approaches. Here, we present a basic method for the purification of the endogenous Par-6/aPKC protein complex, which plays a central role in orchestrating asymmetric cell divisions in the developing nervous system of Drosophila. The procedure can be subdivided into the following steps: acquisition of sufficient starting material, complex stabilization by crosslinking (optional), purification of the protein complex by immunoprecipitation, separation of the isolated material on a polyacrylamide gel, sample preparation for mass spectrometry, and sample analysis. The protocol can easily be adapted to different affinity-tagged or endogenous protein complexes of interest.
authors.affiliation: IMBA-Institute of Molecular Biotechnology, Vienna, Austria.
authors.forename: Christoph
authors.initials: C
authors.lastname: Jüschke
authors.forename: Jürgen A
authors.initials: JA
authors.lastname: Knoblich
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D002455:Cell Division; D003432:Cross-Linking Reagents / Q000494:pharmacology; D015509:Developmental Biology / Q000379:methods*; D029721:Drosophila Proteins / Q000032:analysis / Q000302:isolation & purification*; D004331:Drosophila melanogaster / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006863:Hydrogen-Ion Concentration; D047468:Immunoprecipitation; D013058:Mass Spectrometry / Q000379:methods*; D010449:Peptide Mapping; D025941:Protein Interaction Mapping; D011493:Protein Kinase C / Q000378:metabolism
chemical_list: D003432:Cross-Linking Reagents; D029721:Drosophila Proteins; D011493:Protein Kinase C