The ensemble of expressed proteins in a given cell is organized in multiprotein complexes1,2. The identification of the individual components of these complexes is essential for their functional characterization. The introduction of the 'tandem affinity purification' (TAP) methodology substantially improved the purification and systematic genome-wide characterization of protein complexes in yeast1,3,4. The use of this approach in higher eukaryotic cells has lagged behind its use in yeast because the tagged proteins are normally expressed in the presence of the untagged endogenous version, which may compete for incorporation into multiprotein complexes. Here we describe a strategy in which the TAP approach is combined with double-stranded RNA interference (RNAi)5,6 to avoid competition from corresponding endogenous proteins while isolating and characterizing protein complexes from higher eukaryotic cells. This strategy allows the determination of the functionality of the tagged protein and increases the specificity and the efficiency of the purification.
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This study was supported by the European Molecular Biology Organization (EMBO) and by the Bundesministerium für Bildung und Forschung (BMBF), BioFuture grant no 0311862.
The authors declare no competing financial interests.
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Forler, D., Köcher, T., Rode, M. et al. An efficient protein complex purification method for functional proteomics in higher eukaryotes. Nat Biotechnol 21, 89–92 (2003). https://doi.org/10.1038/nbt773
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