An efficient tandem affinity purification procedure for interaction proteomics in mammalian cells
作者： Tilmann Bürckstümmer , Keiryn L Bennett , Adrijana Preradovic , Gregor Schütze , Oliver Hantschel
DOI: 10.1038/NMETH968
关键词: Protein G 、 Biochemistry 、 Molecular biology 、 Affinity label 、 Affinity chromatography 、 FLAG-tag 、 Biology 、 Peptide 、 Tandem affinity purification 、 Proteome 、 Proteomics
摘要: Tandem affinity purification (TAP) is a generic two-step protocol that enables the isolation of protein complexes under close-to-physiological conditions for subsequent analysis by mass spectrometry. Although TAP was instrumental in elucidating yeast cellular machinery, mammalian cells method suffers from low overall yield. We designed several dual-affinity tags optimized use and compared efficiency each tag to conventional tag. A based on G streptavidin-binding peptide (GS-TAP) resulted tenfold increase protein-complex yield improved specificity procedure. This allows were hitherto not amenable less starting material, leading higher success rates enabling systematic interaction proteomics projects. Using well-characterized Ku70-Ku80 complex as an example, we identified both core elements well new candidate effectors.
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