FXR Transactivation Assay Protocol

Direct FXR activity was evaluated using the LanthaScreen™ TR-FRET Farnesoid X Receptor Coactivator Assay kit (Cat# PV4833, ThermoFisher Scientific). Briefly, prepare a 12-point 100× dilution series of GDCA (Cat#IG2290, Solarbio), CDCA (Cat#IC0300, Solarbio), GW4064 (Cat# HY-50108, MCE) and GUDCA (Cat#IG0840, Solarbio) in a 96-well plate by serial dilution, respectively. Dilute each 100× serial dilution to 2× using Complete Coregulator buffer G. Then, the 2× serial dilutions were mixed with FXR-LBD-glutathione S-transferase fusion protein, fluorecein-SRC2-2 coactivator peptide and Lantha-screen Tb anti-GST antibody (Cat# PV4833, ThermoFisher Scientific, 1:1500) in the 384-well assay plate. Mix the 384-well plate and the TR-FRET signal was evaluated in a Multi-Mode Microplate Reader (Varioskan Flash, Thermo Fisher). Calculate the TR-FRET ratio by dividing the emission signal at 520 nm by the emission signal at 495 nm. Generate a binding curve by plotting the emission ratio vs. [ligand].

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Tang B., Tang L., Li S., Liu S., He J., Li P., Wang S., Yang M., Zhang L., Lei Y., Tu D., Tang X., Hu H., Ouyang Q., Chen X, & Yang S. (2023). Gut microbiota alters host bile acid metabolism to contribute to intrahepatic cholestasis of pregnancy. Nature Communications, 14, 1305.

Publication 2023

Lantha-screen Tb anti-GST antibody Varioskan Flash

Anti antibody Assay Buffer Cdca Dilutions Fret Glutathione s transferase Gw4064 Ligand Peptide Protein

Corresponding Organization :

Other organizations : Xinqiao Hospital, Army Medical University, First People's Hospital of Foshan

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Variable analysis

independent variables

GDCA (Cat#IG2290, Solarbio)
CDCA (Cat#IC0300, Solarbio)
GW4064 (Cat# HY-50108, MCE)
GUDCA (Cat#IG0840, Solarbio)

dependent variables

TR-FRET ratio (emission signal at 520 nm divided by the emission signal at 495 nm)

control variables

FXR-LBD-glutathione S-transferase fusion protein
Fluorecein-SRC2-2 coactivator peptide
Lantha-screen Tb anti-GST antibody (Cat# PV4833, ThermoFisher Scientific, 1:1500)
Complete Coregulator buffer G

positive controls

None explicitly mentioned

negative controls

None explicitly mentioned

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