Protocol for single-molecule pull-down of fluorescently tagged oligomers from cell lysates

Mutations in intrinsically disordered proteins drive the irreversible formation of pathological aggregates, a hallmark of neurodegenerative diseases. Here, we present a protocol to pull down fluorescently tagged proteins to characterize their basal oligomeric states. We describe steps for transfection and cell lysis, single-molecule slide preparation and pull-down, and oligomer dissolution. This protocol enables visualization of protein oligomers with single-molecule resolution. In addition, differences in oligomerization may provide insight on condensation or aggregation propensity in differing mutated or cell stress conditions. For complete details on the use and execution of this protocol, please refer to Djaja et al.1 [Display omitted] •Isolation and pull-down of basal protein oligomers from cell lysates•Dissolution of protein oligomers to provide insight on oligomer formation•Detailed steps for slide preparation and assembly for single-molecule pull-down•Basal protein oligomerization to provide insight on protein condensation Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Mutations in intrinsically disordered proteins drive the irreversible formation of pathological aggregates, a hallmark of neurodegenerative diseases. Here, we present a protocol to pull down fluorescently tagged proteins to characterize their basal oligomeric states. We describe steps for transfection and cell lysis, single-molecule slide preparation and pull-down, and oligomer dissolution. This protocol enables visualization of protein oligomers with single-molecule resolution. In addition, differences in oligomerization may provide insight on condensation or aggregation propensity in differing mutated or cell stress conditions.