| Assay Method Information | |
| | Thyroid Hormone Receptor Binding Assay |
| Description: | The ligand binding domain of thyroid hormone receptor alpha and the ligand binding domain of thyroid hormone receptor beta with GST tag were purchased from Invitrogen. Biotin-SRC2-2 coactivator peptide was purchased from Sangon Biotech, Europium anti-GST and Streptavidin-D2 was from Cisbio. Five microliters of 4× compound serial dilution was added in the 384-well plate, white, low volume, round-bottom assay plate. Five microliters of TRβ LBD (2 nM) or TRα LBD (4 nM) in 50 mM Tris-HCl (pH7.4) with 100 mM NaCl, 1 mM EDTA, 50 mM KF, 1 mM DTT, 1 mM MgCl2, 10% glycerol, 0.01% NP-40, 0.1% BSA (reaction buffer) was mixed with 10 microliters of 400 nM Biotin-SRC2-2 (2×) and anti-GST Eu (1:200) (2×) and 50 nM streptavidin-d2 (2×) in reaction buffer in the same assay plate. Centrifuge the assay plates at 1000 g for 1 min and incubate 1 hr at RT, protected from light. Then read the plate at wavelengths of 665 nm and 615 nm on Envision 2104 plate reader. Calculate EC50 by fitting % Activity values and log of compound concentrations to nonlinear regression (dose response—variable slope) with Graphpad 5.0. |
| Affinity data for this assay | |
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