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

Résumé fourni par la source

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

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