CHD1L Enzyme Inhibition Assay

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.