Purification of Drosophila Protein Complexes for Mass Spectrometry
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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.
