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| | Fluorescence Polarization Assay |
| Description: | Peptides and the ERα LBD were diluted in 1× PBS buffer [140 mM NaCl, 2.7 mM KCl, 10 mM K2HPO4, and 2 mM KH2PO4 (pH 7.4)]. Peptide concentrations were determined by the absorbance at 492 nm, using a fluorescein extinction coefficient of 83000. Fluorescence polarization assays were conducted in 96-well round-bottom black opaque plates (Costar) using 2-fold serial dilutions of the ERα LBD, to final protein concentrations from 10 to 0.0098 μM, in PBS containing 0.1 mM DTT, 20 μM 17β-estradiol, 0.04 mg/mL BSA, and 100 nM fluorescein-labeled peptide. Solutions were incubated in the dark for 30 min at room temperature before being read on a PerkinElmer Fusion plate reader using a 485 nm fluorescein excitation filter and a 535 nm emission filter with a polarizer. |
| Affinity data for this assay | |
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