Single-cluster pull-down assay for visualization and characterization of nanoclusters and condensates.

Biomolecular condensates regulate cellular organization, but early assembly intermediates remain difficult to resolve. Here, we present single-cluster pull-down (SiCluP), a fluorescence microscopy assay to selectively immobilize nanoclusters under defined experimental conditions onto antibody-functionalized PEG surfaces for TIRF imaging and quantitative single-cluster analysis. We describe steps for surface preparation, nanocluster assembly within the flow chamber, selective immobilization, and time-resolved measurements. Using FUS as an example, SiCluP enables the quantification of assembly kinetics, relative cluster size, and molecular exchange at the single-cluster level. For complete details on the use and execution of this protocol, please refer to Ge et al.1.