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PathHunter® Nuclear Hormone Receptor Protein:Protein Interaction Assay Platform

PathHunter protein interaction assay detects binding of an agonist or antagonist to the ligand binding domain of the nuclear hormone receptor (NHR) of interest and the triggering of a functional response. Direct measurement of the response utiizes the specific interaction of the NHR to co-activator/ corepressor protein, which is the last step before gene transcription. Ligand binding induces NHR activation, translocation, and co-activator interaction.


Nuclear Receptor Protein:Protein Assay Principle

Pathhunter protein-protein Nuclear Hormone Platform Schematic
Enzyme Fragment Complementation occurs between two β-galactosidase (β-gal)  components: the ProLink tag is on the C-terminus of the NHR of interest; while the Enzyme Acceptor (EA) fragment is attached to SRCP (steroid response co-activator protein). These fragments interact only when in close proximity, forming active β-gal enzyme that converts substrate to detectable signal.

Measure Compound Efficacy and Potency Differences

GR NHRPRO

GR NHRNUC

Nuclear Translocation Assay Agonist
NHRPRO GR assay (left panel) shows almost complete inhibition of the Dexamethasone response in the presence of Mifipristone, indicating that Mifepristone acts on the target of interest by blocking co-activator recruitment.  In contrast, GR Nuclear Translocation (NHRNUC) assay (right panel) shows no inhibition after addition of Mifepristone. This allows you to study mechanism of action to identify compounds that inhibit co-activator interaction. 

PathHunter Nuclear Hormone Assay Platform Features & Benefits

  • Shorter assay time (6 hours)
  • Measures NHR activation, translocation and coactivator interaction
  • Employs full length NHRs, making data more biologically relevant
  • Applicable to NHRs localized both in the cytoplasm and nucleus
  • Amenable to high-throughput screening
  • Simple plate-treat-read format