Envision microplate analyzer

Anti-GST-coated acceptor beads were used to capture the GST-fusion FXR-LBD whereas the biotinylated-SRC-1 peptide was captured by the streptavidin donor beads. Upon illumination at 680 nm, chemical energy is transferred from donor to acceptor beads across the complex streptavidin-Donor/Src-1-Biotin/GSTFXR-LBD/Anti-GST-Acceptor and a signal is produced. The assay was performed in white, low-volume, 384-well Optiplates (PerkinElmer) using a final volume of 25 μL containing final concentrations of 10 nM of purified GST-tagged FXR-LBD protein, 30 nM biotinylated Src-1 peptide, 20 μg/mL anti-GST acceptor beads acceptor beads and 10 μg/mL of streptavidin donor bead (PerkinElmer). The assay buffer contained 50 mM Tris (pH 7.4), 50 mM KCl, 0.1% BSA, and 1 mM DTT. The stimulation times with 1 μL of tested compound (dissolved in 50% DMSO/H₂O) were fixed to 30 min at room temperature. The concentration of DMSO in each well was maintained at a final concentration of 4%. After the addition of the detection mix (acceptor and donor beads) the plates were incubated in the dark for 4 h at room temperature and then were read in Envision microplate analyzer (PerkinElmer).

Activation of FXR was determined by Alpha Screen Technology in a Coactivator Recruitment Assay. Anti-GST-coated acceptor beads were used to capture the GST-fusion FXR-LBD, whereas the biotinylated-SRC-1 peptide was captured by the streptavidin donor beads. Upon illumination at 680 nm, chemical energy is transferred from donor to acceptor beads across the complex streptavidin-donor/SRC-1-biotin/GSTFXR-LBD/anti-GST-acceptor and a signal is produced. The assay was performed in white, low-volume, 384-well Optiplates (PerkinElmer) using a final volume of 25 μL containing final concentrations of 10 nM of purified GST-tagged FXR-LBD protein, 30 nM biotinylated SRC-1 peptide, 20 μg/mL anti-GST acceptor beads, and 10 μg/mL of streptavidin donor bead (PerkinElmer). The assay buffer contained 50 mM Tris (pH 7.4), 50 mM KCl and 1 mM DTT. The stimulation times with 1 μL of tested compound (dissolved in 50% DMSO/H₂O) were fixed to 30 min at room temperature. The concentration of DMSO in each well was maintained at a final concentration of 2%. After the addition of the detection mix (acceptor and donor beads), the plates were incubated in the dark for 3 h at room temperature and then were read in an Envision microplate analyzer (PerkinElmer).

Anti-GST-coated acceptor beads were used to capture the GST-fusion FXR-LBD, whereas the biotinylated-SRC-1 peptide was captured by the streptavidin donor beads. Upon illumination at 680 nm, chemical energy is transferred from donor to acceptor beads across the complex streptavidin-donor/Src-1-biotin/GSTFXR-LBD/anti-GST-acceptor and a signal is produced. The assay was performed in white, low-volume, 384-well Optiplates (PerkinElmer) using a final volume of 25 μL containing final concentrations of 10 nM of purified GST-tagged FXR-LBD protein, 30 nM biotinylated Src-1 peptide, 20 μg/mL anti-GST acceptor beads acceptor beads, and 10 μg/mL of streptavidin donor bead (PerkinElmer). The assay buffer contained 50 mM Tris (pH 7.4), 50 mM KCl, 0.1% BSA, and 1 mM DTT. The stimulation times with 1 μL of tested compound (dissolved in 50% DMSO/H₂O) were fixed to 30 min at room temperature. The concentration of DMSO in each well was maintained at a final concentration of 4%. After the addition of the detection mix (acceptor and donor beads), the plates were incubated in the dark for 4 h at room temperature and then were read in an Envision microplate analyzer (PerkinElmer).