NUCLEAR PROTEIN PRODUCT SOLUTIONS

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.

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.

Nuclear Protein Translocation Assay Principles

Nuclear protein 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.

Nuclear translocation assays allow for the identification, screening, lead optimization, and 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. Example PathHunter signaling pathway assays shown here measure (A.) TORC1 or TORC2, (B.) p53, and (C.) SREBP2 translocation from the cytosol to the nucleus. The cell lines have been engineered to express the two complementing EFC β-galactosidase (β-gal) fragments within different cellular compartments. The translocating protein is tagged with the small EFC enzyme donor (ED) fragment, while the larger EFC enzyme acceptor (EA) fragment is located in the nucleus. Activation of the pathway by a ligand (small molecule, peptide, or biologic) triggers the ED-tagged protein that is being measured to translocate to the nucleus and complement with the EA to form a fully active β-gal enzyme. The enzyme then hydrolyzes a substrate to generate a chemiluminescent signal that can be easily measured on any standard luminescence reader.

NHR Protein Interaction Assay Principle

NHR protein interaction assays detect the association of the NHR with a coactivator or corepressor accessory protein. The cells have been engineered to express the ED fragment of the β-gal enzyme fused to the NHR (that is located in the nucleus), and the EA fragment fused to the accessory protein (SRC protein). When the NHR is engaged by its small molecule ligand, it forms a complex with the accessory protein, forcing complementation of the ED and EA fragments and creating active β-gal. When provided with a substrate, a chemiluminescent signal is produced.

Measure Activation and Resulting Nuclear Translocation of Transcription Factors

Activation-induced nuclear translocation of transcription factors. PathHunter nuclear translocation cells (Cat. No. 93-0876C3) expressing FOXO3 (Forkhead Box O3; transcription factor) were stimulated with wortmannin for 2 hours leading to the translocation of FOXO3 from the cytoplasm to the nucleus. EC₅₀ of 13.8 nM and signal-to-background (S:B) of 6.7.

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.

Measure Ligand-Induced Association of NHRs with their Accessory Proteins

Obtain quantitative data showing ligand-induced protein interaction between NHRs and their accessory proteins. A. PathHunter RARα cells (Cat. No. 93-0325C2) were stimulated with 9-cis-Retanoic acid for 6 hours, which lead to the interaction of RARα with its accessory protein. EC₅₀ of 8.5 nM and S:B of 5.2. B. PathHunter LXRα cells (Cat. No. 93-0389C2) were stimulated with T0901317 for 6 hours, which lead to the interaction of RARα with its accessory protein. EC₅₀ of 257.2 nM and excellent S:B of 34.6.

Nuclear Translocation Assay Targets

NHR Nuclear Translocation and Protein Interaction Targets