| Assay Method Information | |
| | Biochemical Assay |
| Description: | Active Moz protein was expressed as N-terminal fusion protein with a His6 tag in BL21 E. coli cells. Protein purification was performed via nickel-immobilized metal ion affinity chromatography followed by gel filtration. To determine the inhibition of Moz activity by the test compounds, assay reactions were conducted in a volume of 8 μL in 384-well low volume assay plates. The reactions were performed in assay buffer (100 mM Tris-HCl, pH 7.8, 15 mM NaCl, 1 mM EDTA, 0.01% Tween-20, 1 mM Dithiothreitol, and 0.02% m/v chicken egg white albumin). Reactions were set up with 0.4 μM Acetyl coenzyme A (AcCoA), 50 nM N-terminal histone H4 peptide (sequence SGRGKGGKGLGKGGAKRHRKV-GGK-biotin), 10 nM MOZ enzyme, and an acetyl-lysine specific antibody (final dilution 1:10000). 11-point dilution series of the compounds of the invention were prepared in DMSO; a volume of 100 nL was transferred using a pin tool into assay plates containing substrates, before adding enzyme to start the reaction. Positive (no compound) and negative (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 minutes at room temperature. An additional 4 μL of assay buffer containing AlphaScreen Protein A acceptor beads and Streptavidin donor beads (PerkinElmer, Waltham, Mass.) to a final concentration of 4 μg/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. I |
| Affinity data for this assay | |
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