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Detection of Protein–Protein Interactions Using Tandem Affinity Purification

  • Ian Goodfellow
  • Dalan BaileyEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1177)

Abstract

Tandem affinity purification (TAP) is an invaluable technique for identifying interaction partners for an affinity tagged bait protein. The approach relies on the fusion of dual tags to the bait before separate rounds of affinity purification and precipitation. Frequently two specific elution steps are also performed to increase the specificity of the overall technique. In the method detailed here, the two tags used are protein G and a short streptavidin binding peptide; however, many variations can be employed. In our example the tags are separated by a cleavable tobacco etch virus protease target sequence, allowing for specific elution after the first round of affinity purification. Proteins isolated after the final elution step in this process are concentrated before being identified by mass spectrometry. The use of dual affinity tags and specific elution in this technique dramatically increases both the specificity and stringency of the pull-downs, ensuring a low level of background nonspecific interactions.

Key words

Protein–protein interactions Tandem affinity purification TAP tagging Affinity purification Affinity tags Proteomics Protein purification 

Notes

Acknowledgments

This work was supported by a Wellcome Trust senior fellowship awarded to I.G. and a Research Fellowship from the Pirbright Institute and the University of Birmingham awarded to D.B.

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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Division of Virology, Department of Pathology, Addenbrooke’s HospitalUniversity of CambridgeCambridgeUK
  2. 2.School of Immunity and InfectionUniversity of BirminghamBirminghamUK

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