Tandem Ubiquitin Binding Entities (TUBEs)
Tandem ubiquitin binding entities or TUBE technology is based on the ability to harness the strength of multiple UBDs (Ubiquitin Binding Domains), circumventing the need for immunoprecipitation of overexpressed epitope-tagged ubiquitin or the use of ubiquitin antibodies. Antibody based ubiquitin proteomics is expensive for large scale studies, and, moreover, ubiquitin antibodies sold by most suppliers are notoriously non-selective, leading to artifacts.
LifeSensors has developed technology to specifically isolate polyubiquitylated proteins from cell lysates and tissues. TUBEs are tandem ubiquitin binding domains (UBDs) that bind poly-ubiquitin with Kds in the nanomolar range. TUBEs have also been demonstrated to protect ubiquitylated proteins from both deubiquitylation and proteasome-mediated degradation, even in the absence of inhibitors normally required to block such activities.
Advantages of Choosing LifeSensors’ TUBEs (Tandem Ubiquitin Binding Entities)
- Cost effective compared to alternative technologies
- Kds in the high nanomolar range
- Protection of ubiquitylated substrate
- Wide variety of applications spanning beyond the TPD industry
Our Tandem Ubiquitin Binding Entities have transformed the ubiquitin community due to the powerful ability to selectively capture polyubiquitylated proteins. In addition to development of TUBEs that bind to all types of polyubiquitin chains (pan-TUBEs), LifeSensors has developed chain selective TUBEs that bind selectively to K48 or K63, or linear (M1) polyubiquitylated proteins. The latter are sold as K48 HF TUBEs, K63 TUBEs and M1 TUBEs, respectively. Utilizing pan-selective and chain-selective TUBEs with nanomolar affinity, LifeSensors has developed a variety of assays and services utilizing TUBE technology. Currently, TUBEs are being used by LifeSensors to develop a HTS that elevates PROTAC efficiency aiding TPD (Targeted Protein Degradation) based therapeutics.
Quickly distinguishing true hits from false positives, developing structure-activity relationships, and establishing rank order potency from purified enzymes to cellular models are key steps to success. LifeSensors brings together the tools and expertise necessary to overcome the many pitfalls in the E3 ligase field.
High-throughput tools for monitoring both polyubiquitylation and degradation of a target protein. Measuring ubiquitylation of a protein in a plate-based format accelerates PROTAC-based drug discovery. We have developed several platforms that enable rapid, quantitative monitoring of in vitro as well as cellular ubiquitylation.
Molecular glues (MGs) perform the same function as PROTACs. Molecular glues have a small molecular weight, discovered by traditional screens. Potent and selective glues can be designed with simple chemistry. LifeSensors provides a plethora of tools to discover molecular glues.
LifeSensors’ TUBEs bind to all polyubiquitin chains with 1-10 nM affinity, overcoming major problems common to mass spectrometry proteomics. The combination of the TUBE-based affinity technology and targeted mass spectrometry is the most powerful way to detect the alternations in PMTs and identify signatures for research and biomarkers.
TAMRA-TUBE 2 has a single TAMRA fluorophore (Exc. = 540 nm, Emm. = 578 nm) attached to the fusion tag of TUBE 2 (Cat. No. UM202). Since the fluorophore is attached to the tag, it does not affect binding of the tandem ubiquitin binding domains to polyubiquitin chains allowing for utilization of imaging techniques to study ubiquitination.
TUBEs, such as UM501M are commonly used for isolation and pulling down of ubiquitinated proteins, allowing for a range of applications such as Mass Spec Proteomics, Imaging, or more traditional western blot assays. The below manual describes a detailed procedure for using UM501M for the purposes of pulling down ubiqutinated protiens.