The ubiquitin fluorophore assay utilizes a ubiquitin molecule bound to a quenched fluorophore (such as Ub-Rhodamine, Ub-AMC, or Ub-VME listed below). After the DUB digestion takes place, the fluorophore is released and fluoresces, which is detected via a plate reader. This is the simplest and most straight forward high throughput assay to measure DUB activity. Ubiquitin Rhodamine (Ub-Rho) is a quenched, fluorescent substrate for DUBs, especially ubiquitin C-terminal hydrolases. Cleavage of the amide bond between the C-terminal glycine of ubiquitin and rhodamine results in an increase in rhodamine fluorescence at 535 nm (Exc. 485 nm).
Lifesensors has a multitude of various fluorescent substrates and DUBs to be used in these assays. These DUB assays utilize physiological substrates, polyubiquitin isopeptide chain-selective substrates, and several proteins attached to the C-terminus of ubiquitin that report a fluorogenic signal upon cleavage by DUBs. These substrates make up a large suite of assay technologies available to our customers. No other company provides such a wide variety of tools for HTS and physiological assays for characterizing compounds and establishing a selectivity panel. LifeSensors has expressed, purified, and characterized ~35 DUBs for HTS and compound profiling and has established DUB-centric compound libraries to provide standards for monitoring the inhibition of your compound or characterizing your DUB.