PathHunter® Nuclear Translocation Assays

PathHunter Nuclear Translocation Assays measures movement of proteins from the cytosolic compartment to the nuclear compartment, using an easy-to-use, chemiluminescent readout that does not require microscopy. The Enzyme Fragment Complementation (EFC) technology is used to build this assay. The target protein is tagged with the enzyme donor (ED), and the enzyme acceptor (EA) is confined to the nuclear compartment, using a nuclear localization tag. When the target protein, such as a transcription factor is activated and it translocates to the nucleus, it brings the ED and EA into close proximity and creates an active β-galactosidase enzyme. This active enzyme is able to hydrolyze the substrate and create chemiluminescent light, detectable on any plate-reader. This approach enables you to create a highly specific, non-transcriptional assay for almost any protein that is known to translocate to the nucleus, such as FOXO3, NRF2, XBP1, p53 and many others. Use these cells lines for screening, lead optimization or even testing for compound toxicity!

Technology Principle

Elucidation of NRF2 Translocation Using PathHunter Protein Translocation Assay PlatformNRF2 [Nuclear factor (erythroid-derived 2)-like 2], a transcription factor, and its downstream target genes play an important role in cellular anti-oxidant defense.

Measure specific translocation of NRF-2 into the nucleus

Keap1-NRF2 Stimulated CDDO Methyl Ester U20S Cells

PathHunter U2OS Keap1-NRF2 cells were plated in a 384-well plate and stimulated with CDDO Methyl Ester. Signal was detected using the PathHunter® Detection Kit (93-0001) according to the recommended protocol.

Features & Benefits

  • Simple pipette-and-read protocols
  • Multiple formats – one-step, HTS-ready cell line
  • Broad applicability
  • Simple chemiluminescent detection