| Assay Method Information | |
| | p300 and CBP Protein Activity Assay |
| Description: | Reagent:Reaction buffer: 25 mM HEPES, pH 7.5, 25 mM NaCl, 0.025% CHAPS, 0.025% BSA, 0.5% DMSO.Ligand:Histone H4 (1-21) K5/8/12/16Ac-GG-Biotin.Standard Reaction Conditions:5 nM p300-GST, 50 nM peptide ligand.Reaction Procedure:1. 2.5×BRD was added to the wells of a reaction plate, except for control wells without BRD to which buffer was added instead.2. Compounds in 100% DMSO were delivered into the BRD mixture by acoustic technology (Echo550; nanoliter range). The plate was spun down. The mixture was incubated at room temperature for 30 min.3. 5×peptide ligand was delivered. The plate was spun down. The mixture was incubated at room temperature for 10 min.4. 5×HTRF detection mixture was delivered. The plate was spun down and the mixture was incubated in the dark for 2 h.5. A measurement of HTRF was performed with an Envision reader (Ex/Em=320/615, 665 nm).6. A HTRF ratio was calculated as follows: [em 665 nm/em 615 nm]*10000. |
| Affinity data for this assay | |
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