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.

Influence of fluorescent compounds on the signal of ubiquitin–AMC and the LanthaScreen™ DUB Substrate 
 

Figure 2. Influence of fluorescent compounds on the signal of ubiquitin–AMC and the LanthaScreen™ DUB Substrate 

Due to the TR-FRET readout, the LanthaScreen™ deconjugating assays are ideally suited for screening compound libraries and examining the potency and selectivity of inhibitors (Figure 2.) in either endpoint or kinetic assays (Figure 3.).

Endpoint and kinetic data readouts for the LanthaScreen™ DUB Substrate



Figure 3. Endpoint and kinetic data readouts for the LanthaScreen™ DUB Substrate

Ordering Information

Sku Name Size Price Qty
PV4443 LanthaScreen® Tb-DUB Substrate 10 µg USD 429.30
PV4444 LanthaScreen® Tb-DUB Substrate 100 µg USD 2,828.08