| Assay Method Information | |
| | Rac Activation AlphaScreen Assay (RacI AS) |
| Description: | AlphaScreen assays were performed in 96-well microplates in a final reaction volume of 60 μL. Recombinant His-Rac1, recombinant GST-PBD (PAK Binding Domain), donor and acceptor beads (PerkinElmer), and inhibitors were incubated in exchange buffer (20 mM Tris pH 7.5, 50 mM NaCl, 1 mM MgCl2, 1 mM EDTA, 500 nM GTPyS (guanosine 5′-[Y-thio]triphosphate)) at 37° C. Readings were performed on a Tecan M 1000 pro microplate reader after 1 hour. Data was analyzed using the GraphPad Prism software (GraphPad Software, Inc.), and IC50 (dose leading to 50% disruption of complex) was calculated from the dose-response curves. |
| Affinity data for this assay | |
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