Development of a high-content imaging assay for screening compound aggregation

Aggregated compounds can promiscuously and nonspecifically associate with proteins resulting in either false inhibition or activation of many different protein target classes. We developed a high-content imaging assay in a 384-well format using fluorescently labeled target proteins and an Operetta cell imager to screen for compound aggregates that interact with target proteins. The high-throughput assay can not only directly detect the interaction between compound aggregators and the target of interest, but also determine the critical aggregation concentration (CAC) of a given promiscuous small molecule. •We developed a high-content imaging assay to screen aggregated compounds.•This assay is applicable for directly detecting the interaction of target protein and aggregated compounds.•This assay can determine the critical aggregation concentration of any given compounds.•This assay can be used to scout HTS assay buffer conditions with GFP.