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.
Bimolecular affinity purification (BAP) Mass spectrometry Tandem affinity purification Ubiquitin acceptor site Ubiquitination Ubiquitin-like proteins (UBL)
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Burckstummer T, Bennett KL, Preradovic A et al (2006) An efficient tandem affinity purification procedure for interaction proteomics in mammalian cells. Nat Methods 3:1013–1019PubMedCrossRefGoogle Scholar
Puig O, Caspary F, Rigaut G et al (2001) The tandem affinity purification (TAP) method: a general procedure of protein complex purification. Methods 24:218–229PubMedCrossRefGoogle Scholar
Rigaut G, Shevchenko A, Rutz B et al (1999) A generic protein purification method for protein complex characterization and proteome exploration. Nat Biotechnol 17:1030–1032PubMedCrossRefGoogle Scholar
Maine GN, Gluck N, Zaidi IW, Burstein E (2009) Bimolecular affinity purification (BAP): tandem affinity purification using two protein baits. Cold Spring Harb Protoc 11:1–7Google Scholar
Maine GN, Li H, Zaidi IW, Basrur V, Elenitoba-Johnson KS, Burstein E (2010) A bimolecular affinity purification method under denaturing conditions for rapid isolation of a ubiquitinated protein for mass spectometry analysis. Nat Protoc 5:1447–1459PubMedCrossRefGoogle Scholar
Witze ES, Old WM, Resing KA, Ahn NG (2007) Mapping protein post-translational modifications with mass spectrometry. Nat Methods 4:798–806PubMedCrossRefGoogle Scholar
Schenborn ET, Goiffon V (2000) Calcium phosphate transfection of mammalian cultured cells. Methods Mol Biol 130:135–145PubMedGoogle Scholar
Nielsen ML, Vermeulen M et al (2008) Iodoacetamide-induced artifact mimics ubiquitination in mass spectrometry. Nat Methods 5:459–460PubMedCrossRefGoogle Scholar
Denis NJ, Vasilescu J, Lambert JP et al (2007) Tryptic digestion of ubiquitin standards reveals an improved strategy for identifying ubiquitinated proteins by mass spectrometry. Proteomics 7:868–874PubMedCrossRefGoogle Scholar
Li H, Wittwer T, Weber A et al (2012) Regulation of NF-κB activity by competition between RelA acetylation and ubiquitination. Oncogene 31:611–623PubMedCentralPubMedGoogle Scholar