Affinity purification of ribosomes to access the translatome.
We describe ribosome affinity purification (RAP), a method that allows rapid purification of ribosomes and associated messages from the yeast Saccharomyces cerevisiae. The method relies on the expression of protein A tagged versions of the ribosomal protein Rpl16, which is used to efficiently recover endogenously formed ribosomes and polysomes from cellular extracts with IgG-coupled spherical microbeads. This approach can be applied to profile reactions of the translatome, which refers to all messages associated with ribosomes, with those of the transcriptome using DNA microarrays. In addition, ribosomal proteins, their modifications, and/or other associated proteins can be mapped with mass spectrometry. Finally, application of this method in other organisms provides a valuable tool to decipher cell-type specific gene expression patterns.
Halbeisen
Regula E
RE
Institute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, ETH Zurich, Wolfgang-Pauli-Strasse 10, CH-8093 Zurich, Switzerland.
Scherrer
Tanja
T
Gerber
André P
AP
D002458:Cell Fractionation / Q000379:methods; D002846:Chromatography, Affinity / Q000379:methods*; D040081:Protein Array Analysis / Q000379:methods; D016601:RNA-Binding Proteins / Q000302:isolation & purification; D012269:Ribosomal Proteins / Q000302:isolation & purification*; D012270:Ribosomes / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000302:isolation & purification*
D016601:RNA-Binding Proteins; D012269:Ribosomal Proteins; D029701:Saccharomyces cerevisiae Proteins