LanthaScreen™ Tb Deconjugation Assay Reagents
Deconjugating enzymes (DUBs, Ubiquitin-like (UBL) cleaving enzymes) specifically cleave peptide or isopeptide bonds at the ubiqutin or UBL C-terminus. These proteases are believed to play both “housekeeping” functions, by maintaining pools of active ubiquitin, and regulatory functions, by rescuing specific target proteins from degradation by the proteasome via removal of ubiquitin from the target protein. The LanthaScreen™ Tb deconjugating assay tools provide a sensitive and general method for assaying deconjugating enzyme activity with excellent Z´-factor values.
Figure 1. Mechanism of the LanthaScreen™ deconjugation assay
The LanthaScreen™ Tb DUB Substrate, and the corresponding De-SUMO and De-NEDD8 Substrates, consist of an N-terminal yellow fluorescent protein (YFP) fusions to ubiquitin (or UBLs) with a short C-terminal extension containing an engineered cysteine residue. This engineered cysteine residue is labeled with a LanthaScreen™ terbium chelate to create the TR-FRET based deconjugation substrate. In the absence of a DUB, the LanthaScreen™ Tb DUB Substrate remains intact and exhibits a high degree of FRET. When the substrate is cleaved at the ubiquitin C-terminus by the enzyme, the FRET signal decreases (Figure 1.). Similar assays have been developed for the UBL cleavage enzymes.
Figure 3. Endpoint and kinetic data readouts for the LanthaScreen™ DUB Substrate
To access enzymes for use in our biochemical proteasome assays, please visit
Boston Biochem, Inc.