Tandem Ubiquitin Binding Entities (TUBEs)

Traditional strategies for characterizing ubiquitylated proteins often require 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 using Tandem Ubiquitin Binding Entities (TUBEs).

TUBEs are tandem ubiquitin binding domains (UBDs) that bind tetra-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.

Our technology has 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.

Our assays include:

  • E3 ELISA Assays  – high-throughput system for monitoring E3 ligase activity
  • TR-FRET E3 Assays  – fluorescence-based high-throughput assay system for screening compound libraries against E3 ligase activity
  • UbiTest  – a TUBE-based pull-down method that isolates total cellular ubiquitylated proteins. Subsequently, samples are treated with pan-selective DUBs to remove polyubiquitin chains. The target protein is identified by its native molecular weight and analyzed and quantified by immunoblotting. UbiTest is one of the most sensitive methods available to quantify ubiquitylation levels of proteins in vivo.
  • UbiQuant  – the most sensitive and accurate method for determining the concentration of total ubiquitin (poly + mono) in cell and tissue lysates as a means of monitoring the effects of compounds such as PROTACs, E3 ligase inhibitors, DUB inhibitors, and proteasome inhibitors on cellular proteins.
  • UbiQuant S ELISA and AlphaLISA  – enable accurate determination of substrate ubiquitylation for monitoring the effects of various treatments on patterns of cellular substrate ubiquitylation.
  • UbiQuant Ultra –the latest member of the UbiQuant family of assays, it entails plates coated with high affinity TUBEs, maximizing the likelihood that the ubiquitylated portion of the POI will be captured and quantified.

 

Our services utilizing TUBE technology include ubiquitin E3 ligase screening and profiling, TUBE-based mass spec ubiquitin proteomics, PROTAC drug discovery, and biomarker assay development.