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E3 Ligase Activity Assay

E3 Ligase Activity Assay

E3 ubiquitin ligase is an enzyme that plays a key role in ubiquitin proteasome pathway. Aberrant E3 ligases have been implicated in several diseases and are widely recognized as attractive targets for drug discovery. Therefore, the analysis of E3 ligase activity have a great significance in both biochemical studies and drug discovery efforts.

The detection of E3 ligase activity often utilizes its auto-ubiquitination ability, which is a characteristic exists on almost all E3 ligase. Here we provide several methods to determine the E3 ligase activity in vitro:

  • Western blot-based E3 ligase activity assay

E3 ubiquitin ligase performs its auto-ubiquitination by modifying specific lysine residues in the ligase. Western Blot-based E3 ligase activity assay is a traditional technology that uses specific antibodies for analysis of ubiquitination protein isolated.

  • Enzyme-Linked Immunosorbent Assay (ELISA)-based E3 ligase activity assay

ELISA based assay is another classical method to test protein for ubiquitin activity. This method facilitates ubiquitylation of a known or putative E3 ligase enzymes followed by Western blot analysis. Moreover, ELISA is used for in vitro quantitative measurement of ubiquitin in serum, plasma, tissue homogenates, cell lysates, cell culture supernatant and other biological fluids.

  • Tandem Ubiquitin Binding Entities (TUBEs)-based E3 ligase activity assay

TUBEs bind selectively to polyubiquitin chains due to the nanomolar affinity, enabling the detection of polyubiquitin chains in the presence of mono-ubiquitin. This assay is an approach for characterizing and quantitating E3 ligase activity in a rapid and cost effective manner.

  • Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET)-based E3 ligase activity assay

TR-FRET detection is a way to quantitate the binding of TUBEs to the polyubiquitin chains as the product of the E3 ligase reaction. FRET is a technique that relies on proximity dependent transfer of resonance energy from a donor fluorophore to an acceptor fluorophore. TR-FRET utilizes the lanthanide chemistry that provides E3 ligase activity assay in qualitatively and quantitatively. With spatial resolution, and high sensitivity for donor/acceptor pairs, this method avoids the background fluorescence from sample components such as buffers, proteins, chemical compounds and cell lysate.

The advantages of our E3 ligase activity assay services:

  • Advanced technology platforms
  • Strict and standardized process
  • Professional team and extensive experience
  • Highly reliable results and analysis
  • Short turn-around time and competitive price

Profacgen offers services from ligase expression to screen and development for a one-stop PROTAC development services. We promise to work closely with customers, please contact us for more information.


1. Marblestone JG, LaRocque JP, Mattern MR, Leach CA. (2012). Analysis of ubiquitin E3 ligase activity using selective polyubiquitin binding proteins. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research, 1823(11), 2094–2097. doi:10.1016/j.bbamcr.2012.06.013
2. Zhao Q, Liu L, Xie Q. (2011). In Vitro Protein Ubiquitination Assay. Plant Signalling Networks, 163–172. doi:10.1007/978-1-61779-809-2_13

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