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DUB-Detector™

simple fluorescent assay to detect deubiquitinating enzyme activity

DUB-Detector™ Deubiquitination Assay provides a simple solution for screening activity of deubiquitination enzymes (DUBs) from cell extracts or purified recombinant proteins in human, mouse and rat systems. The assay contains a fluorescent ubiquitin substrate that when processed by the cysteine protease class of DUBs releases a fluorescent signal that is proportional to the amount of enzymatic activity. For added convenience, HeLa nuclear extract and a DUB inhibitor are included as positive and negative controls. This assay can be used for either endpoint or kinetic analysis of enzyme activity, or to screen for DUB inhibitors.

For complete assay details, click the Method tab below. To learn more about deubiquitination enzymes or to see assay data, click the DUB Info tab below; kit manuals can be downloaded under the Documents tab.

 
Name Format Cat No. Price  
DUB-Detector™ 1 x 96 rxns 40110 $495 Buy Now

Deubiquitination Enzymes

Ubiquitination is a reversible, post-translational modification in which the 76 amino acid polyubiquitin peptide is added to proteins by the sequential action of three enzymes: E1 ubiquitin-activiating enzyme, E2 ubiquitin-conjugating enzyme and E3 ubuiquitin ligase. Protein ubiquitination is a highly dynamic process and the removal of ubiquitin from proteins is now considered to be equally important for protein regulation.

The large group of enzymes that are responsible for the removal of ubiquitin from proteins are known as deubiquitination enzymes (also known as DUBs, deubiquitylating enzymes or ubiquitin deconjugating enzymes). The human genome encodes nearly 100 deubiquitinating enzymes, making them the largest family of enzymes in the ubiquitin system. DUBs are responsible for ubiquitin precursor processing, ubiquitin recycling, trimming of ubiquitin chains, as well other diverse roles in cell growth and differentiation, devlopment, DNA damage, disease pathways, transcriptional regulation and chromatin remodeling.

Deubiquitination enzymes can be divided into five families: ubiquitin-specific proteases (USPs), ubiquitin C-terminal hydrolases (UCHs), ovarian tumour proteases (OTUs), Machado-Joseph disease protein domain proteases (MJDs) and JAB1/MPN/MOV34 metalloproteases (JAMM). The first four families are classified as cysteine proteases and comprise the majority of deubiquitinating enzymes. The last family, JAMM, bind zinc and therefore are classified as metalloproteases. The cysteine hydrolases specifcally cleave ubiquitin substrates with the general structure UB1-72-Leu73-Arg74-Gly75-Gly76-X, where X can be any small thiol, amine, ubiquitin molecule or even another protein.

USPs are the largest family of deubiquitination enzymes and they are also the group with the largest size variation (50-300 kDa). These high molecular weight DUBs can process ubiquitin precursors, remove ubiquitin from protein conjugates and disassemble long ubiquitin chains. In contrast to the USPs, the UCHs are relatively small enzymes (<30 kDa) that catalyze the removal of peptides and small molecules from the C-terminus of ubiquitin. The table below provides a sample of deubiquitination enzymes and their associated biological function.

Deubiquitination Enzyme Biological Function
USP3, USP7, USP16, USP21, USP22, MYSM1, BRCC36 Chromatin remodeling
USP1, USP3, USP28 DNA damage
USP7, USP16, USP19, USP28, CYLD Cell proliferation
 

DUB-Detector™ Data

 

Kinetic analysis of DUB activity in HeLa nuclear extract
 
Figure 1: Kinetic analysis of DUB activity in HeLa nuclear extract.

HeLa nuclear extracts were assayed at 0.313, 0.625 and 1.25 µg per reaction in the presence or absence of 1 µM inhibitor for 20 minutes. Following the incubation, Fluorescent Substrate (100 nM) was added to each well and the fluorescent intensity was measured every two minutes. Data shown are the results from duplicates.

 
Endpoint analysis of DUB activity in HeLa nuclear extract
 
Figure 2: Endpoint analysis of DUB activity in HeLa nuclear extract.

The DUB-Detector Assay was used to assay activity of HeLa nuclear extracts at 0.313, 0.625 and 1.25 µg per reaction in the presence or absence of 1 µM inhibitor for 20 minutes. Following the incubation, Fluorescent Substrate (100 nM) was added to each well and the reaction proceeded for 60 minutes before the fluorescent intensity was measured with an excitation wavelength of 485 nm and an emission wavelength of 535 nm. The fold change in fluorescence was plotted. Data shown are the results from duplicates.

 
Recombinant USP16 activity
 
Figure 3: Recombinant USP16 deubiquitination activity.

The DUB-Detector Assay was used to assay activity of recombinant USP16 at 12.5, 25 and 50 pM per reaction in the presence or absence of 100 nM inhibitor for 20 minutes. Following the incubation, Fluorescent Substrate (100 nM) was added to each well and fluorescent intensity was immediately measured with an excitation wavelength of 485 nm and an emission wavelength of 535 nm. The fluorescent intensity was measured every minute with a total reaction time of 60 minutes. Data shown are the results from duplicates.

Why use DUB-Detector™?

  • Fluorescent assay can be detected with an excitation wavelength of 485 nm and an emission wavelength of 535 nm
  • Complete assay with optimized buffers for enhanced enzymatic acitivity
  • Includes both positive control extract and a universal DUB inhibitor
  • Fast procedure can be completed in less than 1 hour
  • Great for either kinetic or endpoint analysis
  • Quickly screen for DUB inhibitors
 

DUB-Detector™ Method

Despite the differences in their cellular roles and molecular sizes, the deubiquitinating enzymes all appear to hydrolzye their substrates through a common mechanism. Active Motif's DUB-Detector Assay uses a universal ubiquitin substrate to detect enzymatic activity or screen for potential inhibitors. The fluorescent substrate is based on a C-terminal derivative of ubiquitin which is hydrolyzed by the cysteine protease class of enzymes to release a fluorescent signal proportional to the amount of enzyme activity. The fluorescence can be detected using a microplate reader with excitation of 485 nm and emission of 535 nm. For added convenience, the assay also includes HeLa nuclear extract and an inhibitor to all classes of deubiquitinating enzymes as positive and negative controls.

Flow chart of the DUB-Detector Assay for detecting deubiquitinating enzyme activity
 
Figure 1: Flow chart of the DUB-Detector™ Method.

Sample containing deubiquitinating enzyme activity is combined with complete assay buffer and transferred to a 96-well half area plate. Fluorescent substrate is added to each well and the plate is read using an excitation wavelength of 485 nm and emission wavelength of 535 nm. (Click image to enlarge.)

Contents & Storage

DUB-Detector™ contains a universal Fluorescent Substrate, Assay Buffer AM2, 1 M DTT, 96-well half area plate, and reagent reservoirs. Inhibitor and HeLa nuclear extract are provided as negative and positive controls. Reagent storage conditions vary from room temperature to -80°C, see manual for details. All reagents are guaranteed stable for 6 months when stored properly.