Low volume non binding surface 384 well plates

All reactions were carried out using low volume non-binding surface 384-well plates (Corning Inc., Corning NY). 100 nM cat-CHD1L or fl-CHD1L and 200 nM c-Myc DNA or mononucelosome (Active Motif, Carlsbad, CA) were added to a buffer containing 50 mM Tris pH 7.5, 50 mM NaCl, 1 mM DTT, 5% glycerol, and the reaction was initiated by the addition of 10 μM ATP (New England Biolabs, Ipswich, MA) to a total volume of 10 μL and incubated at 37 °C for 1 h. ATPase activity was measured by adding 500 nM of Phosphate Sensor (Life Technologies, Carlsbad, CA) measuring excitation 430 and emission 450 immediately on an Envision plate reader (PerkinElmer, Hopkinton, MA). An inorganic phosphate standard curve was used to convert the fluorescence to [Pi], and enzyme kinetics were determined using GraphPad Prism (San Diego, CA).

CHD1L enzyme inhibition assay was performed as described previously.^{11 (link)} All reactions were carried out using low volume non-binding surface 384-well plates (Corning Inc., Corning, NY). 800 nM cat-CHD1L, 200 nM mononucleosome (Active Motif, Carlsbad, CA), and various concentrations of inhibitors were preincubated at 37 °C for 10 min in 1x buffer containing 50 mmol/L Tris pH 7.5, 50 mmol/L NaCl, 5 mmol/L MgCl₂, 2 mmol/L DTT, and 5% glycerol. The reaction was initiated by the addition of 10 μmol/L ATP (New England Biolabs, Ipswich, MA) to a total volume of 10 μL and incubated at 37 °C for 1 hour. ATPase activity was measured by adding 500 nmol/L of Phosphate Sensor (ThermoFisher) measuring excitation (430 nm) and emission (450 nm) immediately on an Envision plate reader (PerkinElmer). Background signal was determined by using all assay components excluding the enzyme.