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Tandem Affinity Purification Combined with Mass Spectrometry to Identify Components of Protein Complexes

  • Peter Kaiser
  • David Meierhofer
  • Xiaorong Wang
  • Lan Huang
Part of the Methods in Molecular Biology™ book series (MIMB, volume 439)

Abstract

Most biological processes are governed by multiprotein complexes rather than individual proteins. Identification of protein complexes therefore is becoming increasingly important to gain a molecular understanding of cells and organisms. Mass spectrometry—based proteomics combined with affinity-tag-based protein purification is one of the most effective strategies to isolate and identify protein complexes. The development of tandem-affinity purification approaches has revolutionized proteomics experiments. These two-step affinity purification strategies allow rapid, effective purification of protein complexes and, at the same time, minimize background. Identification of even very low-abundant protein complexes with modern sensitive mass spectrometers has become routine. Here, we describe two general strategies for tandem-affinity purification followed by mass spectrometric identification of protein complexes.

Keywords

tandem-affinity purification (TAP) His-Bio tag mass spectrometry protein complex identification in-vivo cross-linking MudPIT 

Notes

Acknowledgments

We thank Christian Tagwerker and Cortnie Guerrero for their contributions in developing the protocols described here and Karin Flick for the figure design. Work in the laboratory of P. Kaiser is supported by grants from NIH (RO1GM-66164, R21CA113823) and the California Breast Cancer Research Program (11NB-0177). L. Huang acknowledges support from NIH (GM074830) and the DOD (PC-041126). D. Meierhofer is supported by an Erwin Schroedinger Fellowship (FWF J2665).

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Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Peter Kaiser
    • 1
  • David Meierhofer
    • 1
  • Xiaorong Wang
    • 2
  • Lan Huang
    • 2
  1. 1.Department of Biological Chemistry, College of MedicineUniversity of California IrvineIrvineUSA
  2. 2.Department of Physiology and Biophysics and Department of Developmental and Cell BiologyUniversity of California IrvineIrvineUSA

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