A parallel affinity purification method for selective isolation of polyubiquitinated proteins.
We developed a parallel affinity purification (PAP) procedure, in which ubiquitinated proteins are purified from the cells that coexpress two affinity-tagged ubiquitins by sequential use of affinity chromatography specific to each tag. In contrast with previous procedures using a single affinity-tagged ubiquitin, the PAP eliminates highly abundant ubiquitin monomers and monoubiquitinated proteins to selectively enrich proteins bearing both affinity-tags, or poly- and multiubiquitinated proteins. Accordingly, it would serve as a powerful method to facilitate mass-spectrometric identification of ubiquitinated proteins.
Ota
Kazuhisa
K
Department of Computational Biology, Graduate School of Frontier Sciences, University of Tokyo, Kashiwa, Japan.
Kito
Keiji
K
Iemura
Shun-ichiro
S
Natsume
Tohru
T
Ito
Takashi
T
D002846:Chromatography, Affinity / Q000379:methods*; D025821:Polyubiquitin / Q000378:metabolism*; D011506:Proteins / Q000302:isolation & purification* / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D054875:Ubiquitination
D011506:Proteins; D025821:Polyubiquitin