Deubiquitylases (DUBs) are proteases that are responsible for the release of ubiquitin or ubiquitin chains from target proteins in vivo.  This activity requires the cleavage of an isopeptide bond between the terminal glycine of ubiquitin and the e-amino group of a lysine residue in the target.  Until now, all high throughput compatible deubiquitylase assays have measured the cleavage of a linear amide or peptide bond between ubiquitin and either a small fluorophore (e.g. AMC, Rhodamine110) or a reporter protein (CHOP assays).  LifeSensors’ new IQF-DiUb? substrates are the first commercially available deubiquitylase substrates that permit continuous monitoring of isopeptide bond cleavage in a homogeneous format.  This is achieved by incorporating a fluorescent dye on one ubiquitin and an appropriate fluorescence quenching dye on the second, resulting in an internally quenched fluorescence (IQF) molecule.  Cleavage of the isopeptide bond linking the ubiquitin subunits results in an increase in fluorescent signal when the quencher and fluorophore are separated.  In order to minimize perturbation of ubiquitin’s structure, the dyes are attached to specific sites within the ubiquitin molecule and not at the N- or C- termini.  Since some labeling sites may affect recognition by any given DUB, we have created a panel of different diubiquitin molecules within each linkage class using several different labeling sites, e.g. we offer six different versions of our K48 linked diubiquitin.  We recommend that you start with the survey panel of diubiquitins in order to determine which version of a particular linkage class gives the optimal results under your assay conditions in terms of sensitivity, signal to background, and turn over by your DUB.

The generation of ubiquitin isopeptidase assays using physiologically relevant substrates represents a major advancement in the study of this important group of cellular enzymes.

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