An efficient protein complex purification method for functional proteomics in higher eukaryotes.
The ensemble of expressed proteins in a given cell is organized in multiprotein complexes. 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 yeast. 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) 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.
Forler
Daniel
D
European Molecular Biology Laboratory, Heidelberg, Germany.
Köcher
Thomas
T
Rode
Michaela
M
Gentzel
Mark
M
Izaurralde
Elisa
E
Wilm
Matthias
M
D000818:Animals; D002478:Cells, Cultured; D002846:Chromatography, Affinity / Q000379:methods*; D004331:Drosophila melanogaster / Q000737:chemistry / Q000378:metabolism; D005057:Eukaryotic Cells / Q000737:chemistry / Q000502:physiology; D046911:Macromolecular Substances; D013058:Mass Spectrometry / Q000379:methods*; D046912:Multiprotein Complexes; D011506:Proteins / Q000235:genetics / Q000302:isolation & purification*; D020543:Proteome / Q000737:chemistry* / Q000235:genetics; D040901:Proteomics / Q000379:methods*; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods
D046911:Macromolecular Substances; D046912:Multiprotein Complexes; D011506:Proteins; D020543:Proteome; D012330:RNA, Double-Stranded; D011994:Recombinant Proteins