| Assay Method Information | |
| | THR/RXR Heterodimer Assay for Thyroid Hormone Receptor Agonist Screening |
| Description: | Test compounds were prepared as 10 mM DMSO stock solutions. The stock solution (45 uL) was transferred to a 384-well assay plate, and 3-fold, 10-point dilutions were performed by transferring 15 μL of the compound solution into 30 μL DMSO using TECAN (EVO200) liquid handler. The compound solutions (200 nL, serially diluted) and the positive control triiodothyronine (T3) (100 nL) were transferred to an assay plate using ECHO550. Next, H6-THR-α (150.64 uM, 10 μL) or H6-THR-β (32.57 uM, 10 μL) in binding buffer (50 mM HEPES, pH 7.0, 1 mM DTT, 0.05% NP40, 0.2 mg/mL BSA) was mixed with retinoid X receptor alpha (RxRα) (146.76 uM, 10 μL) in binding buffer, and transferred to the 384-well assay plate containing T3 or compound solution. After incubation at 37° C. for 30 min, biotin-GRIP1 peptide (3262.1 uM, 10 μL) in binding buffer and 5% DMSO was added to the 384-well assay plate and incubated at 37° C. for 30 min. A solution (10 μL) containing europium-conjugated anti-hexa(His) antibody (0.625 uM) and APC-conjugated streptavidin (1.18 uM) in buffer (50 mM Tris, pH 7.4, 100 mM NaCl, and 0.2 mg/mL BSA) was then added to the 384-well assay plate and incubated at 25° C. for 60 min. The assay plate was read using Envision (PerkinElmer), using T3 as the positive control for both THR-β/RXR-α and THR-α/RXR-α activity. DMSO was used as the negative control. Compound activity for the THR-β/RXR-α and THR-α/RXR-α assays were normalized to T3 activity for each assay run. THR-β selectivity was calculated by dividing the normalized THR-β/RXR-α compound activity by the normalized THR-α/RXR-α compound activity. |
| Affinity data for this assay | |
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