| Assay Method Information | |
| | Scintillation Proximity Assay (SPA) |
| Description: | The p300 HAT domain (residues 1287-1666) was expressed and purified with an N-terminal His tag from Escherichia coli cells. The expressed protein was purified by Ni2+ affinity, followed by anion exchange chromatography. Appropriate fractions were pooled and buffer exchanged into 20 mM Hepes pH 7.5, 150 mM NaCl, and 1 mM TCEP. Compounds of interest solubilized in DMSO were stamped in a Greiner black 384-well plate in a 10-point duplicate dose response using an Echo 550 (Labcyte). p300-HAT domain purified in-house (aa 1287-1666) was diluted to 6 nM in reaction buffer (50 mM Tris pH 8.0, 100 mM NaCl, 1 mM DTT, 0.069 mM Brij-35, 0.1 mM EDTA, 0.1 mg/mL BSA), combined with 4.14 uM AcCoA (Sigma-Aldrich) and 0.46 uM 3H-AcCoA (PerkinElmer), and 12.5 uM added to each well and incubated for 30 min at RT. Reactions were initiated with 12.5 uL 2 uM biotinylated H3(1-21) peptide (New England Peptide) and run for 1 hr at RT, then quenched with 20 uL stop solution (200 mM Tris pH 8.0, 200 mM EDTA, 2M NaCl, 160 uM anacardic acid). 35 uL of the reaction volume was transferred to a 384-well streptavidin FlashPlate (PerkinElmer) using a Bravo liquid handler (Velocity 11) and incubated for 1.5 hr at RT. Plates were aspirated, washed with 95 uL wash buffer (15 mM Tris pH 8.5, 0.069 uM Brij-35), aspirated, sealed, and scintillation counts read on a Topcount (PerkinElmer). |
| Affinity data for this assay | |
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