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Isopeptidase Assays

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Novel assays for characterizing Ubiquitin and Ubiquitin-like isopeptidase activity

CHOP REPORTER ASSAYS
ROBUST   -   SENSITIVE   -   EASY TO USE

CHOP-Reporter technology offers a rapid and robust in vitro assay for ubiquitin and ubiquitin-like isopeptidase activity.

Introduction

The concept behind the Ub/Ubl-CHOPReporter Deconjugation Assay is the fusion of Ub/Ubl with reporter enzyme. The nature of this fusion renders the reporter enzyme catalytically inactive. Upon cleavage of the Ub/Ubl-fusion by an isopeptidase, the activated, free reporter enzyme acts upon its substrate and generates a fluorescent signal. Thus, in the coupled assay, the signal generated by cleavage of the reporter enzyme’s substrate is a quantitative measure of isopeptidase activity. CHOP and CHOP2 platforms utilize different reporter enzymes making multiplexing possible.

CHOP: A Revolutionary & Superior Assay

Previous Ub/Ubl isopeptidase assays relied on the fusion of small chemical adducts to the C-terminus of the Ub/Ubl. In many cases this molecule is not recognized by the isopeptidase of interest.

The CHOP assay platforms utilize protein fusions which are more physiologically relevant and can be recognized by isopeptidases that do not recognize other substrates.

One example is the COP9 signalsome. The COP9 signalosome (CSN) is a highly conserved protein complex that was originally described as a repressor of light-dependent growth and transcription in Arabidopsis. CSN is a multi-subunit protease that regulates the activity of cullin-RING ligase (CRL) families of ubiquitin E3 complexes by removing NEDD8 from the E3 ligase. The CSN plays a crucial role in multiple cellular processes including the regulation of DNA damage repair, cell cycle progression, and gene expression. Until now there has not been a method for measuring the activity of CSN in a high-throughput manner. The NEDD8-CHOP2 assay is the first HT method for detecting CSN activity and will revolutionize the study of CSN activity. Refer to the figures 2 & 3 in the Gallery, Related download section.

About the assay

The CHOP & CHOP2-Reporter Assay consists of target fused to a reporter enzyme, as well as a separate reagent substrate for the reporter enzyme. Upon conjugation to target, the reporter is rendered catalytically inactive. Following cleavage of the target-reporter system by the isopeptidase, the free (and now active) reporter subsequently acts upon its substrate. Thus, in the coupled assay, the signal generated by cleavage of the reporter enzyme’s substrate is a quantitative measure of isopeptidase activity.

 

The CHOP2 reporter system is compatible with many detergents and has significantly lower background than the CHOP, allowing longer kinetic reads (> 6hours)

 

Benefits

  • Rapid and robust readout of isopeptidase activity
  • Reporter substrates are non-radioactive
  • Amenable to HTS and miniaturization for cost effective screening
  • Detection of deconjugation from a macromolecular conjugate protein
  • Readout does not require excitation in the UV range (reducing false positives)

Applications

  • Demonstration of novel deubiquitinase activity
  • High throughput screening (HTS) for either agonists or antagonists of deubiquitinase activity

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