Developing novel tetraubiquitin substrates and AlphaLISA technology to provide next generation deubiquitinase assays

Ubiquitination is the modification of cellular proteins with ubiquitin. It is a dynamic and reversible physiological process, affecting the stability and function of proteins, with deubiquitinating enzymes (DUBs) removing attached ubiquitin from proteins.

Most DUB assays currently use fluorescent substrates derived from monoubiquitin, which may not adequately mimic the natural substrates that a DUB acts upon in vivo.

This application note presents novel biochemical DUB assays that use TUBEs to bind to tetraubiquitin substrates of at least three natural linkages. These assays, combined with Revvity’s AlphaLISA^™ technology, allow for the measurement of protein interactions and large protein complexes in a no-wash, homogeneous assay. The note includes data illustrating the utility of these assays and demonstrating the specificity of certain DUBs for tetraubiquitin substrates of different linkages.

For research use only. Not for use in diagnostic procedures.