| Assay Method Information | |
| | AlphaScreen Assay |
| Description: | The protocol for the CBX7 AlphaScreen was as follows: a standard alpha buffer (50 mM HEPES, 150 mM NaCl, 0.1% w/v BSA, 0.01% w/v Tween20) at pH 8.0 and make up two stock solutions at 2× final concentration in the alpha buffer. Solution A contains Human His6-CBX7 is used with biotinylated-H3K27me3 (residues 20-34), at 100 nM and 50 nM respectively. Solution B contains 20 nM streptavidin donor beads and 20 nM nickel acceptor beads. For 384 well assay formats, 10 uL of solution A is added to each well of the assay plate and the plate is spun at 1000 rpm for 30 seconds. 100 nL of experimental compounds from stock plates are delivered by robotic pin transfer using a Janus Workstation (PerkinElmer), allowing the compounds to interact with CBX7 binding prior to assay measurement, followed by another spin and an incubation room temperature. Finally, 10 uL of solution B is added to each well, the plate is spun again and then incubated at room temperature. |
| Affinity data for this assay | |
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