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An efficient tandem affinity purification procedure for interaction proteomics in mammalian cells

Nature Methods volume 3, pages 10131019 (2006) | Download Citation

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Abstract

Tandem affinity purification (TAP) is a generic two-step affinity purification 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 the yeast cellular machinery, in mammalian cells the method suffers from a low overall yield. We designed several dual-affinity tags optimized for use in mammalian cells and compared the efficiency of each tag to the conventional TAP tag. A tag based on protein G and the streptavidin-binding peptide (GS-TAP) resulted in a tenfold increase in protein-complex yield and improved the specificity of the procedure. This allows purification of protein complexes that were hitherto not amenable to TAP and use of less starting material, leading to higher success rates and enabling systematic interaction proteomics projects. Using the well-characterized Ku70-Ku80 protein complex as an example, we identified both core elements as well as new candidate effectors.

*Note: In the version of this article initially published online, the GS-TAP tag in Figure 3b was incorrectly identified as a GC-TAP tag, and the email address for material requests (materials@cemm.oeaw.ac.at) was omitted from the methods section. The errors have been corrected for all versions of the article.

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Change history

  • 06 October 2006

    In the version of this article initially published online, the GS-TAP tag in Figure 3b was incorrectly identified as a GC-TAP tag, and the email address for material requests (materials@cemm.oeaw.ac.at) was omitted from the methods section. The errors have been corrected for all versions of the article.

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Acknowledgements

We thank M. Brehme for help with graphical illustrations. We also thank J.-M. Peters (Institute for Molecular Pathology, Vienna), S.P. Jackson (Wellcome Trust/Cancer Research UK Gurdon Institute, Cambridge), M. Mann (Max Planck Institute for Biochemistry, Martinsried) and R. Aebersold (Eidgenoessische Technische Hochschule, Zuerich) for critical reading of the manuscript. We thank M. Planyavsky for preparation of the TAP samples for analysis by mass spectrometry and the entire Superti-Furga laboratory for suggestions. T.B. is supported by a research fellowship (BU 2180/1-1) from the German Research Foundation (DFG). CeMM is supported by the Austrian Academy of Sciences. Work described here was funded by the Austrian Proteomics Platform-II of the GenAU Program of the Austrian Ministry of Education and Research, and by a grant from the Austrian National Bank.

Author information

Affiliations

  1. Research Center for Molecular Medicine (CeMM), Lazarettgasse 19/3, 1090 Vienna, Austria.

    • Tilmann Bürckstümmer
    • , Keiryn L Bennett
    • , Adrijana Preradovic
    • , Gregor Schütze
    • , Oliver Hantschel
    • , Giulio Superti-Furga
    •  & Angela Bauch

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Competing interests

G.S.-F. has received a consultancy salary from the drug discovery/proteomics company Cellzome Inc. as a member of its scientific advisory board.

Corresponding author

Correspondence to Giulio Superti-Furga.

Supplementary information

PDF files

  1. 1.

    Supplementary Fig. 1

    Detailed characterization of the AC- and the GS-TAP-tag.

  2. 2.

    Supplementary Fig. 2

    GS-TAP-Ku70: Biochemical activity and specificity control.

  3. 3.

    Supplementary Fig. 3

    The GS-TAP tag allows for a single-step purification.

Word documents

  1. 1.

    Supplementary Table 1

    Lists of interactors associated with Ku70 and nonspecific interactors as identified my mass spectrometry analysis.

  2. 2.

    Supplementary Methods

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DOI

https://doi.org/10.1038/nmeth968

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