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Bimolecular Affinity Purification: A Variation of TAP with Multiple Applications

  • Petro Starokadomskyy
  • Ezra BursteinEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1177)

Abstract

The identification of true interacting partners of any given bait can be plagued by the nonspecific purification of irrelevant proteins. To avoid this problem, Tandem Affinity Purification (TAP) is a widely used procedure in molecular biology as this reduces the chance of nonspecific proteins being present in the final preparation. In this approach, two different affinity tags are fused to the protein bait. Herein, we review in detail a variation on the TAP procedure that we have previously developed, where the affinity moieties are placed on two different proteins that form a complex in vivo. This variation, which we refer to as Bimolecular Affinity Purification (BAP), is suited for the identification of specific molecular complexes marked by the presence of two known proteins. We have utilized BAP for characterization of molecular complexes and evaluation of proteins interaction. Another application of BAP is the isolation of ubiquitin-like proteins (UBL)-modified fractions of a given protein and characterization of the lysine-acceptor site and structure of UBL-chains.

Key words

Bimolecular affinity purification (BAP) Mass spectrometry Tandem affinity purification Ubiquitin acceptor site Ubiquitination Ubiquitin-like proteins (UBL) 

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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Department of Internal MedicineUT Southwestern Medical CenterDallasUSA
  2. 2.Departments of Internal Medicine and Molecular BiologyUT Southwestern Medical CenterDallasUSA

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