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Assay Method Information |
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| | AlphaScreen Assay (AS) |
| Description: | Each 384-well plate was prepared with 4.75 µL of working assay buffer (50 mM HEPES, 10 mM DTT, 100 mM NaCl, 0.05% Tween029 and 0.1 mg/mL casein, pH 7.4) and 0.25 µL of compound stock (20 mM in DMSO) per well. Both binding partners, BCl-xL and the acceptor beads, and biotinylated BH3 peptide and the donor beads were preincubated for 30 min. 10 µL of the acceptor bead-BCL-xL complex was added to all of the wells and the plates were covered and incubated for 30 min in room temperature. 10 µL of the donor bead-BH3 peptide complex was then added to each well and the plates were again covered and incubated for 4 h at room temperature and then read using a PerkinElmer Fusion Alpha plate reader (Ex680, Em520-620 nM). |
| Affinity data for this assay | |
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