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 polyubiquitin moieties over the single ubiquitin binding associated domain. In addition, TUBEs protect polyubiquitinated proteins from deubiquitination and proteasomal degradation, for detection at relatively low abundance. These properties effectively allow TUBEs to “capture” protein in a polyubiqutinated state. TUBEs are used to isolate, enrich, and identify ubiquitinated proteins from cell and tissue extracts. Magnetic beads coupled to TUBE 2 allow for efficient recovery of polyubiquitinated proteins in a single step without centrifuge. Magnetic TUBEs allow more complete removal of the unbound supernatant, with lower background. Polyubiquitinated proteins can be separated from TUBEs for proteomics or identification by Western blotting.