Prism 5.0 demo

All SPA experiments were performed with Copper His-tag YSi Scintillation proximity beads (PerkinElmer, RPNQ0096). Beads were diluted to 2.5 mg/ml in 150 mM MES-NaOH, pH 6.5, 50 mM NaCl, 20% glycerol, 2 mM TCEP and 0.05% DDM. 100 nM [³H]-ifenprodil was incubated with 800 nM N-terminal His₆-tagged EBP protein in a total volume of 100 μl at 4 °C for 2 h. The solution was added to 100 μl SPA beads and incubated by vigorous shaking at 4 °C in the dark for 2 h. The mixture was loaded into individual wells of 96-well plates. Samples were measured in duplicate at room temperature with a Wallac 1450 MicroBeta plate PMT counter. For the competition assay, the concentration of tamoxifen or U18666A was increased from 0.1 nM to 1 mM. To define the nonspecific binding activity, 400 mM imidazole was added to the wells to compete with the His₆-tag for bead binding. Nonspecific binding was subtracted from each data point. All experiments were performed at least three times and data are presented as mean ± s.d. Data fitting was performed using GRAPHPAD PRISM 5.0 Demo.

Statistical analyses were performed using t-test or ANOVA followed by the Newman-Keuls multicomparison test when appropriate. Statistical significance was set at P < 0.05. Data were analyzed using the GraphPad Prism 5.0 Demo program.