ER Binding Assays
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Competition binding assay is run in a buffer containing 50mM Hepes, pH 7.5, 1.5mM EDTA, 150mM NaCl, 10% glycerol, 1mg/ml ovalbumin and 5mM DTT, using 0.025 μCi per well 3HEstradiol, 10 ng/well ERalpha or ERbeta receptor. Competing compounds are added at 10 different concentrations. Non-specific binding is determined in the presence of 1μM of 17-β Estradiol. The binding reaction (140 μl) is incubated for 4 hours at room temperature, then 70 μl of cold DCC buffer is added to each reaction (DCC buffer contains per 50 ml of assay buffer, 0.75g of charcoal and 0.25g of dextran). Plates are mixed 8 minutes on an orbital shaker at 4°C. Plates are then centrifuged at 3,000 rpm at 4°C for 10 minutes. An aliquot of 120μl of the mix is transferred to another 96-well, white flat bottom plate and 175μl of Wallac Optiphase “Hisafe 3” scintillation fluid is added to each well. Plates are sealed and shaken vigorously on an orbital shaker. After an incubation of ~5 hrs, read plates in a Wallac Microbeta counter. The data is used to calculate an IC50 and % Inhibition at 10μM. The Kd for 3H-Estradiol is determined by saturation binding to ER alpha and ER beta receptors. The IC50 values for compounds are converted to Ki using Cheng-Prusoff equation and the Kd determined by saturation binding assay.
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References:
1. Norman, B. H., Dodge, J. A., Richardson, T. I., Borromeo, P. S., Lugar, C. W., Jones, S. A., Chen, K., Wang, Y., Durst, G. L., Barr, R. J., Montrose-Rafizadeh, C., Osborne, H. E., Amos, R. M., Guo, S., Boodhoo, A. and Krishnan, V. (2006) Benzopyrans are selective estrogen receptor beta agonists with novel activity in models of benign prostatic hyperplasia. J Med Chem. 49, 6155-6157
2. Hilbish, K. G., Breslin, W. J., Johnson, J. T. and Sloter, E. D. (2013) Fertility and developmental toxicity assessment in rats and rabbits with LY500307, a selective estrogen receptor beta (ERbeta) agonist. Birth Defects Res B Dev Reprod Toxicol. 98, 400-415
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