PathHunter nuclear protein assays are based on the proprietary Enzyme Fragment Complementation (EFC) technology to measure the movement of proteins into the nucleus or the protein-protein interactions in the nucleus.
Nuclear Translocation Assay Principles
Nuclear translocation assays allow for the identification, screening, lead optimization, and even safety testing of therapeutics. These assays monitor the activation or inhibition of a particular signaling pathway coupled with the movement of proteins from the cytosolic compartment to the nuclear compartment. In general, 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. Learn about several translocation assays on the nuclear proteins and signaling pathways pages.
NHR Nuclear Translocation Assay Principle
NHR translocation assays detect the binding of small molecules that trigger NHR activation and translocation into the nucleus. The cells have been engineered to express the ED fragment of the β-gal enzyme fused to the NHR (that is located in the cytoplasm) and the EA fragment localized in the nucleus. Ligand binding to the NHR induces the receptor translocation from the cytoplasm to the nucleus, forcing the complementation of the ED and EA fragments and the formation of an active β-gal enzyme. The enzyme then hydrolyzes a substrate to generate a chemiluminescent signal that can be easily measured on any standard luminometer.
Measure Activation-Induced Nuclear Translocation of Transcription Factors
Quantitative data showing binding and ligand-induced translocation on NHRs such as androgen, glucocorticoid, liver X, mineralcorticoid, and progesterone receptors. Monitoring Glucocorticoid Receptor Nuclear Translocation. A. Immunofluorescence analysis of dexamethasone induced Glucocorticoid Receptor translocation into the nucleus using PathHunter CHO-K1 Glucocorticoid Receptor Nuclear Translocation cells. B. Translocation EFC-based assay (Cat. No. 93-0002C2) of the same cells stimulated with dexamethasone for 3 hours and results shown an EC50 of 8.2 nM and excellent signal-to-background of 15.9. PathHunter NHR translocation assays employ full length NHRs providing more biologically relevant data, and shorter assay times allowing for fewer off-target effects.