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

Abstract

Summary: 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 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Keywords

• Single-molecule Assays
• Cell-based Assays
• Molecular Biology