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Enzyme Fragment Complementation Assay Technology

Enzyme Fragment Complementation (EFC) is a patented detection technology based on two recombinant β-galactosidase (β-gal) fragments – a large protein fragment (enzyme acceptor, EA) and a small peptide fragment (enzyme donor, ED). Separately, the β-gal fragments are inactive, but when combined, they form an active β-gal enzyme that hydrolyzes substrate to produce a chemiluminescent signal.

Enzyme Fragment Complementation Assay Platform

Single Technology - Multiple Applications

DiscoverX's proprietary Enzyme Fragment Complementation (EFC) technology offers drug discovery the means to interrogate biomolecular reactions for advancing therapeutic drug screening and development programs. As a robust and reliable assay technology, EFC can be used for both cell-based (PathHunter®, InCELL) and biochemical (HitHunter®) assay formats.

  • An enzymatically amplified signal, resulting in large signal to background ratios and high precision with Z' factors > 0.7
  • Chemiluminescent signal minimizes interference from library compounds, introducing no artificial signal due to non-specific binding of beads or secondary labels
  • Scalable protocols for 96, 384 and 1536-well microplate applications
  • Homogenous, mix-and-read assay protocols - EFC assays do not require washing, centrifugation or filtering
  • Reproducible, low hit rates and low batch-to-batch variation saves reagents and reduces operating costs

PathHunter Cell-based Enzyme Fragment Complementation Assay Platform

The PathHunter cell-based assays can be applied to study a number of events including protein translocation or trafficking, protein-protein interaction, secretion and degradation. This is achieved by tagging the protein of interest with the ProLink (low affinity) or the ProLabel (high affinity) fragment of β-gal enzyme. Depending on the event that is studied, the EA fragment of β-gal is either appended to the second protein (interaction studies), localized to the membrane or cellular compartment (translocation studies) or added exogenously to study protein secretion and degradation.
Learn more about the PathHunter cell-based assay platform > 

HitHunter Biochemical Enzyme Fragment Complementation Assay Platform

HitHunter biochemical assays can be used to study multiple applications including compound-target engagement, measure and rank compound potencies, quantify ligand levels, perform functional screens, identify novel compounds, and more.  This is accomplished by either tagging the protein or ligand of interest with the ED fragment of β-gal and adding the EA fragment exogenously.
Learn more about the HitHunter biochemical assay platform >

InCELL Cellular Protein-binding Assay Platform

Employing EFC, the catalytic domain of the protein of interest is tagged with enhanced ProLabel (ePL). Complementation between the ePL and EA occurs only when compounds bind to the catalytic domain and stabilizes the protein of interest, increasing its half-life relative to the unbound protein.
Learn more about the InCELL assay platform >

Utilize EFC to Study Multiple Target Classes


2nd Messenger



Kinase Binding



SH2 Recruitment






Transcription Factor



Nuclear Translocation