Ubiquitin-Proteasome Pathway Assays
Target-specific Assays

Deubiquitination Assays (DUB assays)

The ubiquitin-proteasome pathway is responsible for the degradation of intracellular proteins as part of the general protein turnover, regulation of gene transcription, and protein abundance in cells as well as quality control. Proteins are being ubiquitylated to mark them for degradation by proteasomes. This process is highly regulated by ubiquitin-ligases and deubiquitinases. Ubiquitin is expressed as a fusion protein precursor that needs to be processed by deubiquitinating enzymes to yield monomers.

20S Proteasome Activity Assay measures the chymotrypsin-like activity by cleaving the fluorescent substrate Suc-LLVY-AMC.

  • Low-scale, large-scale and high-throughput DUB screening options are available
  • Please ask us for deubiquitinase assay development and custom-tailored options
  • Deubiquitinase enzymatic activity is measured with Ubiquitin-AMC fluorescent substrate

List of Proteasome and DUB Assays (23)

Deubiquitination Assay Details

  • Deubiquitinase Assays
  • Assay Setup
Deubiquitinase Assays

Fluorescent Ubiquitin-AMC is a substrate containing a fluorophore, 7-amido-4-methyl coumarin (AMC), that is quenched when ubiquitylated. After cleavage by a deubiquitinase, AMC is released and its fluorescence in the absence and presence of a deubiquitinase inhibitor can be measured.

Assay Setup

Setups: Single-dose screening in duplicates or IC50 value determination with 5 or 10 concentrations. Other screening formats are available upon request.

Controls: No inhibitor (DMSO vehicle) control and for every assay, one target-specific control compound is tested in 10-dose IC50 format.

Turnaround time: 10 business days for standard projects. Expedited scheduling and data delivery can be arranged prior to the commencement of the studies.

Report: The raw data, % enzyme activity and control compound IC50 values will be reported in Excel format for single-dose assays. For IC50 orders, raw data, IC50 values, and curve fitting will be delivered in Excel format. Assay conditions, target, and substrate information are available upon request. Requirements for this information should be noted prior to the commencement of the study.

Screening facility: This assay is performed at our screening facility in Malvern, PA, US.

Compound requirements: In brief, for a standard project, 20 µl of a 10 mM DMSO stock or solid material is needed. Less material is needed for large scale screening. Please refer to our FAQs for information regarding compound preparation and shipping.