TY - JOUR
T1 - Bimolecular affinity purification
T2 - A variation of TAP with multiple applications
AU - Starokadomskyy, Petro
AU - Burstein, Ezra
N1 - Publisher Copyright:
© Springer Science+Business Media New York 2014.
PY - 2014
Y1 - 2014
N2 - 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.
AB - 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.
KW - Bimolecular affinity purification (BAP)
KW - Mass spectrometry
KW - Tandem affinity purification
KW - Ubiquitin acceptor site
KW - Ubiquitin-like proteins (UBL)
KW - Ubiquitination
UR - http://www.scopus.com/inward/record.url?scp=84925546136&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84925546136&partnerID=8YFLogxK
U2 - 10.1007/978-1-4939-1034-2_15
DO - 10.1007/978-1-4939-1034-2_15
M3 - Article
C2 - 24943324
AN - SCOPUS:84925546136
SN - 1064-3745
VL - 1177
SP - 193
EP - 209
JO - Methods in Molecular Biology
JF - Methods in Molecular Biology
ER -