TUBEs: Ubiquitin Affinity Matrix
Tandem Ubiquitin Binding Entities (TUBEs) are powerful reagents for DETECTION and ISOLATION of polyubiquitylated proteins. LifeSensors offer varieties of TUBEs reagents, like Non-selective TUBEs and linkage specific TUBEs with different tags (Biotin, His, FLAG etc.) For more information about TUBEs
Ubiquitin and Polyubiquitylation
Ubiquitin is a small polypeptide that can be conjugated via its C-terminus to amine groups of lysine residues on target proteins. This conjugation is referred to as monoubiquitylation. Additional ubiquitin moieties can be conjugated to this initial ubiquitin utilizing any one of the seven lysine residues present in ubiquitin. The formation of these ubiquitin chains is referred to as polyubiquitylation. The two most well characterized forms of polyubiquitylation occur via linkage at lysine 48 (K48) or lysine 63 (K63). The most prevalent consequence of polyubiquitylation is the proteasome-mediated degradation of the target protein. Polyubiquitylation is a reversible process, however, as these chains are degraded and/or removed by proteases known as deubiquitylases (DUBs). The dynamic nature of this signaling represents a major obstacle to the isolation and functional characterization of polyubiquitylated proteins. For this reason, the ubiquitylation state of many proteins is unknown or poorly characterized.
TUBEs: A Revolution in Polyubiquitin Isolation and Characterization
Traditional strategies for characterization of ubiquitylated proteins often require immunoprecipitation of overexpressed ubiquitin with an epitope tag or the use of ubiquitin antibodies (expensive for large scale studies). Alternatively, isolation of polyubiquitylated proteins can be achieved with certain ubiquitin binding associated domains (UBAs), but these proteins display a low affinity for ubiquitin. Additionally, these strategies require the inclusion of inhibitors of both DUB and proteasome activity to protect the integrity of polyubiquitylated proteins. These conditions could alter cell physiology, which in turn may negatively impact the result or introduce experimental artifact. To overcome these problems, Dr. Manuel Rodriguez and his team at CIC bioGUNE have developed Tandem Ubiquitin Binding Entities (TUBEs). TUBEs are essentially tandem UBAs with dissociation constants for tetra-ubiquitin in the nanomolar range. TUBEs have also been demonstrated to protect proteins from both deubiquitylation and proteasome-mediated degradation, even in the absence of inhibitors normally required to block such activity. The nanomolar affinity of TUBEs for polyubiquitylated proteins allows for high efficiency in isolation and characterization of these proteins from cell lines, tissues and organs.
Non-Selective TUBEs (Pan-TUBEs)
High affinity pan-TUBEs allow for identification and characterization of polyubiquitin proteins by western blotting, as well as isolation of proteins for downstream proteomic studies. Our non-selective TUBEs, TUBE1 and TUBE 2, are derived from different ubiquitin binding domains and as such may exhibit slight differences in their binding to specific polyubiquitylated target proteins. However, these differences are typically inconsequential. In general the binding profiles are very similar. Both TUBE 1 and TUBE 2 bind to all linkages of polyubiquitins.
LifeSensors currently has M1(linear)-, K48-, and K63-linkage selective TUBEs. The promising UBAs are engineered to bind specifically to a particular linkage such as they display 100-10,000-fold higher affinity for their cognate polyubiquitin over all other polyubiquitin chains. These linkage-specific TUBEs are not based on the identity of the UBA itself, but rather on the structural context of the UBA within the TUBE as a consequence of the helical nature of the linkers that separate each domain. The linkage-selective TUBEs are a sensitive and effective tool for detection and isolation of linkage-specific polyubiquitylated proteins in the cell and tissue lysates.