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DiUbiquitin substrates

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Novel DiUbiquitin substrates (DiUb) to measure DUB activity

Protein ubiquitylation is a dynamic and reversible process involving a diverse group of deubiquitylating enzymes (DUBs). These enzymes recognize and cleave the isopeptide bond between ubiquitin moieties. One of the challenges in monitoring DUBs activity is to create a physiologically relevant substrates. 

LifeSensors diubiquitin substrates represent a new class of continuous assay substrates for the cleavage of a true isopeptide bond.  C-terminus of wild type ubiquitin is conjugated in vitro via an isopeptide bond to lysine 48 or lysine 63 of a second ubiquitin molecule and the resultant diubiquitin forms an internally quenched fluorescent FRET pair (IQF) in which the fluorescence quencher is located on one ubiquitin moiety and the fluorescent dye (TAMRA) is located on the other molecule.

The assay is based on the gain in TAMRA fluorescent signal  following a cleavage of the diubiquitin molecule by specific DUBs  leading to separation of the fluorophore from quencher. 

In DiUb assay, diubiquitins are prepared with the energy transfer pairs in different positions (K63-1-2-3-4-5-6 or K48-1-2-3-4-5-6) in order to allow optimization of a given diubiquitin substrate for an individual DUB.  Importantly, the introduction of fluorophore or quencher residue does not affect the native structure of the ubiquitin molecule.

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

Benefits

  • Fast and easy method of determining best DiUb for your DUB
  • Capable of detecting relevant hits missed with traditional substrates such as Ub-RHO110*
  • More biologically relevant substrate compared to traditional mono-ubiquitin derivatives
  • The quencher/fluorophores are NOT located at either the N- or C- terminus, thus preserving the native structure of ubiquitin

 Applications

Download our applications notes to learn more (Related download section on the right of this page)

 

Which DiUb for me?

The placement of the internally quenched fluorescence dyes on each ubiquitin subunit may affect binding and/or turnover of the diubiquitin substrates. For this reason, we have created six variants for each of the currently available IQF-DiUbs with the dyes in different positions.

In order to find which variant works best with your DUB(s), we recommend that you start with one of the DiUb selection panels: the DiUb48 Panel (DU0101) or the DiUb63 Panel (DU0102) . Each panel contains all six variants of the respective (K48/K63) linkage and a control enzyme. If the linkage specificity of your DUB is unknown or if it cleaves both linkages, you can use the DiUb K48/K63 panel (DU0201) which contains all 12 variants.

 

DiUb K-48

50 µg each

DiUb K-63

50 µg each

Panels

DiUb K-11  NEW

50 µg each

 DiUb K48-1 (DU4801)  DiUb K63-1 (DU6301)  DiUb K48 1-6 panel (DU0101)  DiUb K11-4 (DU1104)
 DiUb K48-2 (DU4802)  DiUb K63-2 (DU6302)  DiUb K63 1-6 panel (DU0102)  DiUb K11-6 (DU1106)
DiUb K48-3 (DU4803) DiUb K63-3 (DU6303) DiUb K-48 1-6 & DiUb K-63 1-6 panel (DU0201)  DiUb K11-8 (DU1108)
DiUb K48-4 (DU4804) DiUb K63-4 (DU6304)    
DiUb K48-5 (DU4805) DiUb K63-5 (DU6305)    
DiUb K48-6 (DU4806) DiUb K63-6 (DU6306)    

 


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