| Assay Method Information | |
| | Binding AlphaLISA Assay |
| Description: | The AlphaLISA assay was performed in a 384-well Proxiplate in a total volume of 40 μL. The reaction mixture contained 0.0625 nM 6×His-Mcl-1 (171-327), 0.0625 nM biotinylated-Bim peptide, 10 μg/mL AlphaLISA anti-6×His-AlphaLISA acceptor beads, 40 g/mL AlphaScreen streptavidin donor beads, and serially diluted test compounds in the binding buffer (20 mM Hepes, pH 7.5; 150 mM NaCl); 0.002% Brij 35; 1 mM Dithiothreitol (DTT) Solution; 0.01% BSA). 1,000×test compounds were pre-spotted onto 384-well Proxiplate by Echo 555 Liquid Handler followed by incubation of 5 μl Mcl-1(171-327) for 1 hour. Then 5 μL Bim (51-76) was added and incubated for 2 hours. Five μL AlphaLISA anti-6His-AlphaLISA acceptor beads were then added for 1 hour followed by addition of 5 μL AlphaScreen streptavidin donor beads for 1 hour. The reaction plates were then read on an Envision multimode reader using AlphaScreen settings. |
| Affinity data for this assay | |
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