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| | HTS discovery of chemical inhibitors of anti-apoptotic protein Bfl-1 |
| Description: | The aim is to identify chemical probes of Bfl-1 through a fluorescence polarization assay (FPA) using FITC-Bid BH3 peptide. 1) Dose-response curves contained 10 concentrations of compounds obtained using 2-fold serial dilution. Compounds were serially diluted in 100% DMSO, and then diluted with water to 10% final DMSO concentration. 4 uL compounds in 10% DMSO were transferred into columns 3-22 of Greiner 384-well black small-volume plates (784076). Columns 1-2 and 23-24 contained 4 uL of 10% DMSO. 2) Columns 1-2 were reserved for positive controls and added with 8 uL of assay buffer using WellMate bulk dispenser (Matrix). 3)8 uL of Bfl-1 working solution was added to columns 2-24 using WellMate bulk dispenser (Matrix). Columns 23-24 represent negative control wells. 4) Plates were incubated for 1h at +4oC. 5)8 uL of freshly prepared FITC-Bid working solution was added to the whole plate using WellMate bulk dispenser (Matrix). 6) Plates were incubated for 4h at room temperature protected from direct light. 7) Fluorescence polarization was measured on an Analyst HT plate reader (Molecular Devices, Inc) using fluorescein filters: excitation filter - 485 nM, emission filter # 530 nM, dichroic mirror # 505 nM. The signal for each well was acquired for 100 ms. 8) Data analysis was performed using sigmoidal dose-response equation through non-linear regression. |
| Affinity data for this assay | |
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