| Assay Method Information | |
| | EED A-395 Peptide Competition Assay |
| Description: | For the assay, compounds or nonbiotinylated H3(23-34) peptide were dispensed in assay-ready plates using a three-fold serial dilution from 50 μM to ~850 pM using an Echo 550 Acoustic Liquid Handler (Labcyte). The following buffer was used to set up the binding reactions: 20 mM Tris-HCl pH 7.5, 200 mM NaCl, 0.01% Tween-20, 10 μM DTT, and 0.05% BSA, with the DTT and BSA added fresh before initiating the binding assay. The binding assay was initiated by adding a 10 μl mixture of 1 nM GST-tagged EED, 15 nM biotinylated-histone H3 peptide, and 10 μg/ml each of the streptavidin donor and glutathione acceptor beads to the predispensed compounds. The reactions were then incubated for 1 h at 25 °C in a humidified chamber before detection on the PerkinElmer Envision plate reader using an AlphaLISA/Screen protocol. |
| Affinity data for this assay | |
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