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PathHunter® Enzyme Fragment Complementation Assay Platform


DiscoverX has exploited the versatile features of enzyme fragment complementation (EFC) technology to study specific cell-based protein activities. PathHunter assays are designed using the two inactive fragments of β-galactosidase (β-gal), the enzyme donor (ED) and the enzyme acceptor (EA), which combine to create an active enzyme. 

PathHunter EFC Assay Principle

PathHunter Enzyme Fragment Complementation
PathHunter Enzyme Fragment Complementation Schematic
PathHunter cells are genetically engineered to over-express the two proteins under study, one fused to PK and the second protein fused to EA. Interactions between the two proteins drives complementation between PK and EA, resulting in formation of an active β-gal enzyme that cleaves a substrate to generate chemiluminescent signal.

Features & Benefits

  • Single functional readout – applicable to different GPCRs, NHRs and kinase receptors and families
  • Stable cell lines - increased reliability and reproducibility
  • Full length protein – ideal for large molecule therapeutics and small molecule inhibitors
  • High specificity – tagged protein eliminates background from endogenous proteins
  • High quality reproducible data – superior assay windows &  performance (Z' > 0.6)
  • Simple readout using standard plate readers

Explore Target Biology With Multiple Technology Platforms

Protein:protein Interactions

A  homogenous cell-based assay format to detect in vivo interaction of two proteins where the ProLink fragment is attached to one protein and the EA is attached to the second protein. Active β-gal enzyme is formed only when the two proteins interact with each other. The robust assays are specific, sensitive and highthroughput adaptable and use the EFC technology.

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Protein Translocation

PathHunter cells are genetically engineered to over-express the protein under study fused to PL/PK and the EA localized to one of the following organelles: nucleus, cell membrane, or endosome. Upon compound stimulation of cells, the PL/PK- tagged protein undergoes translocation into the cell compartment in which the EA-is localized. This results in the formation of the active β-gal enzyme which can be detected using a chemiluminescent substrate.

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Protein Degradation

PathHunter protein degradation assays involve a protein of interest tagged with a small ProLink peptide. The assays measure specific ProLink-tagged protein degradation in response to pathway stimulation.

Learn more about PathHunter Protein Degradation Assays >