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

Nathalie Djaja; Sua Myong

doi:10.1016/j.xpro.2023.102716

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

STAR Protoc. 2023 Dec 15;4(4):102716. doi: 10.1016/j.xpro.2023.102716. Epub 2023 Nov 17.

Authors

Nathalie Djaja¹, Sua Myong²

Affiliations

¹ Program in Cell, Molecular, Developmental Biology, and Biophysics, Johns Hopkins University, Baltimore, MD 21218, USA; Department of Biology, Johns Hopkins University, Baltimore, MD 21218. Electronic address: ndjaja1@jhu.edu.
² Department of Biology, Johns Hopkins University, Baltimore, MD 21218; Department of Biophysics, Johns Hopkins University, Baltimore, MD 21218. Electronic address: smyong1@jhu.edu.

Abstract

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.

Keywords: Cell-based Assays; Molecular Biology; Single-molecule Assays.

MeSH terms

Cell Death
Intrinsically Disordered Proteins*
Mutation
Transfection

Substances

Intrinsically Disordered Proteins

Abstract

MeSH terms

Substances

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