| Assay Method Information | |
| | Acetyltransferase Biochemical Assay |
| Description: | o determine the inhibition of KAT enzymatic activity by test compounds, assay reactions were conducted in a volume of 8 uL in 384-well low volume assay plates. The reactions were performed in assay buffer (100 mM Tris-HCl, pH7.8, 15 mM NaCl, 1 mM EDTA, 0.01% Tween-20, 1 mM Dithiothreitol and 0.01% m/v chicken egg white albumin). Reactions were set up with 1 uM Acetyl coenzyme A, 100 nM of full-length recombinant histone labelled by limited biotinylation (KAT6A, KAT6B, KAT7: H3.1, KAT5, KAT8: H4), 10/5/8/40/20 nM of KAT5/KAT6A/KAT6B/KAT7/KAT8 enzyme respectively, a an acetyl-lysine specific antibody (H3.1: Cell Signaling Technology, H4: Abcam). 11-point dilution series of the test compounds were prepared in DMSO; a volume of 100 nL was transferred using a pin tool into assay plates containing substrates, before (AcCoA omitted) control reactions were included on the same plates and received the same amount of DMSO as the compound treated wells. After adding all reagents, the plates were sealed with adhesive seals and incubated for 90 min at room temperature. An additional 4 uL of assay buffer containing AlphaScreen Protein A acceptor beads ug/mL was then added. After incubation for 2 hours the plates were read using an EnVision 2103 multi label plate reader (PerkinElmer) in HTS AlphaScreen mode. |
| Affinity data for this assay | |
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