Ligand binding to nuclear receptors causes conformational changes in the receptor, resulting in a cascade of events, including dissociation of repressor proteins,association of coactivator proteins, and assembly of pol II and other transcriptional factors for activation of target genes. TR-FRET based assays can be developed using the LanthaScreen™ panel of fluorescein-labeled coregulator peptides to investigate conformational changes of nuclear receptors upon ligand binding, either by determining the affinity of ligand- bound receptor for different coregulator peptides, or by identifying additional agonists or antagonists via displacement or recruitment of a specific coregulator peptide. 

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LanthaScreen™ TR-FRET NR Coregulator Interaction Assay Kits contains the reagents necessary to perform an assay to assess the ligand dose dependency for the recruitment of coregulator peptides to nuclear receptor targets.

LanthaScreen™ fluorescent nuclear receptor coregulator peptides contain known interaction motifs and are labeled with fluorescein. Assays using these reagents enable primary or secondary screening of nuclear receptor agonists and/or antagonists.


 

Figure 1. Principle of nuclear receptor agonist-dependent coactivator peptide recruitment. Tb-anti-GST antibody indirectly labels RXR-β by binding to the GST tag on the N-terminus. Binding of the agonist, 9-cis-RA, to RXR-β causes a conformational change that results in an increase in the affinity of RXR-β for fluorescein-D22, a labeled coactivator peptide. The close proximity of the fluorescently labeled coactivator peptide to the Tb-labeled antibody causes an increase in the TR-FRET signal.

 

 

Figure 2. Representative data measuring recruitment of fluorescein-D22 coactivator peptide to RXR-β. Serial dilution of agonist 9-cis retinoic acid, 1 nM RXR β- LBD, 350 nM fluorescein-D22 peptide, and 2 nM Tb-anti-GST antibody. Results for 1-hr, 2-hr, 4-hr, and 6-hr incubations are shown with the corresponding EC50 values. The curves were generated using a sigmoidal dose response (variable slope) equation in GraphPad™ Prism® 4.0.

 

Data

LanthaScreen™ TR-FRET NR Coregulator Interaction Assay Kits contains the reagents necessary to perform an assay to assess the ligand dose dependency for the recruitment of coregulator peptides to nuclear receptor targets.

LanthaScreen™ fluorescent nuclear receptor coregulator peptides contain known interaction motifs and are labeled with fluorescein. Assays using these reagents enable primary or secondary screening of nuclear receptor agonists and/or antagonists.


 

Figure 1. Principle of nuclear receptor agonist-dependent coactivator peptide recruitment. Tb-anti-GST antibody indirectly labels RXR-β by binding to the GST tag on the N-terminus. Binding of the agonist, 9-cis-RA, to RXR-β causes a conformational change that results in an increase in the affinity of RXR-β for fluorescein-D22, a labeled coactivator peptide. The close proximity of the fluorescently labeled coactivator peptide to the Tb-labeled antibody causes an increase in the TR-FRET signal.

 

 

Figure 2. Representative data measuring recruitment of fluorescein-D22 coactivator peptide to RXR-β. Serial dilution of agonist 9-cis retinoic acid, 1 nM RXR β- LBD, 350 nM fluorescein-D22 peptide, and 2 nM Tb-anti-GST antibody. Results for 1-hr, 2-hr, 4-hr, and 6-hr incubations are shown with the corresponding EC50 values. The curves were generated using a sigmoidal dose response (variable slope) equation in GraphPad™ Prism® 4.0.

 

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For Research Use Only. Not for use in diagnostic procedures.