Purification of Drosophila protein complexes for mass spectrometry.
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.
Jüschke
Christoph
C
IMBA-Institute of Molecular Biotechnology, Vienna, Austria.
Knoblich
Jürgen A
JA
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
D003432:Cross-Linking Reagents; D029721:Drosophila Proteins; D011493:Protein Kinase C