PUBCHEM_BIOASSAY: qHTS screen for small molecules that inhibit ELG1-dependent DNA repair in human embryonic kidney (HEK293T) cells expressing luciferase-tagged ELG1. (Class of assay: confirmatory) [Related pubchem assays (depositor defined):AID493107, AID493125]
Inhibition Assay: 384-well plates (Costar #3654) were filled using a liquid handling robot with 20 uL of a mixture of 50 nM of a fluorescein-linked UDP-GlcNAc analog (see Gross et al, 2003), 1-2 uM sOGT, and buffer (20 mM potassium phosphate, pH=7.4 with 500 uM tris(hydroxypropyl)phosphine). The 1249 compound library was serially diluted in DMSO from the 5 mg/ml plates fivefold 3 times, such that 4 different concentrations of compounds were prepared. Compound libraries of the 4 concentrations in duplicated were then transferred to the assay plates using a 100 nL pin array, resulting in a final compound concentration of 25 ug/mL or 70 uM at the highest of the four concentrations, assuming an average compound MW of 350. Using a Perkin Elmer Envision microplate reader, the sample was excited at 480 nm in the vertical plane, and simultaneous emission intensity (535 nm) of the vertical and horizontal polarization planes was measured.