HitHunter® & PathHunter® Enzyme Fragment Complementation Assay Technology

Enzyme Fragment Complementation Assay Platform

HitHunter®

In the biochemical assays, ED is conjugated to ligand of interest (cAMP, cortisol, or cGMP) which competes against free ligand for binding to a binding protein or antibody. In the absence of free ligand, ED-conjugates are captured by the antibody and are unavailable for complementation. In the presence of free ligand, binding protein sites are occupied, leaving ED-conjugate free to complement with EA, forming active EFC β-gal enzyme for substrate hydrolysis to produce a detectable signal.

PathHunter®

The PathHunter® cell-based assays can be applied to study a number of events including protein translocation, 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 in the nucleus (translocation studies) or added exogenously to study protein secretion and degradation.

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 biochemical and cell-based assay formats.

 

 

EFC Capabilities for Drug Discovery

HitHunter®		PathHunter®

• 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

	GPCRs		Kinases		Epigenetics		Pathways		NHRs
	Arrestin Internalization 2nd Messenger		Dimerization Phosphorylation Kinase Binding		Methyltransferases Bromodomains		Transcription Factor Regulated Proteolysis/Ubiquitin		NHR-Protein Interactions Nuclear Translocation

Explore Target Biology With Multiple Technology Platforms

PathHunter® Cell-based Enzyme Fragment Complementation Assay Platform

PathHunter®: EFC whole-cell format is a flexible platform ideal for evaluating protein-protein interactions, secretion, translocation trafficking and degradation.

Learn more about the PathHunter biochemical assay platform >

HitHunter® Biochemical Enzyme Fragment Complementation Assay Platform

HitHunter biochemical assays are designed as homogenous, competitive assays for precise, highly sensitive detection of specific analytes.

Learn more about the HitHunter biochemical assay platform >

InCELL Hunter™ 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.