Based on protein domains known to possess an affinity for ubiquitin, Tandem Ubiquitin Binding Entities (TUBEs) have been developed for the isolation and identification of ubiquitinated proteins. TUBEs display up to a 1000-fold increase in affinity for polyubiquitin moieties over the single ubiquitin binding associated domain (UBA). In addition, TUBEs display a protective effect on polyubiquitinated proteins, allowing for detection at relatively low abundance. These properties effectively “capture” protein in its polyubiquitin state. Agarose coupled TUBEs allows for efficient recovery of polyubiquitinated proteins in a single step. Polyubiquitinated protein can easily be separated from TUBEs for proteomic studies or for identification by western blotting.
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