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 ubiquitylated proteins. TUBEs display up to a 1000-fold increase in affinity for poly-ubiquitin 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" proteins in their polyubiquitylated state. Biotin-TUBE 2 is based on UBA1 from the human RAD23A. Biotin-TUBEs allow for the detection of polyubiquitin and polyubiquitylated proteins by ligand blotting ("far Western") without heating the membrane. This reagent is a superior alternative to traditional polyubiquitin immunodetection techniques, such as anti-ubiquitin IgGs.
