Single‐Molecule Two‐Color Coincidence Detection of Unlabeled alpha‐Synuclein Aggregates
Alexandre Chappard, Craig Leighton, Rebecca S. Saleeb, Kiani Jeacock, Sarah R. Ball, Katie Morris, Owen Kantelberg, Ji‐Eun Lee, Elsa Zacco, Annalisa Pastore, Margaret Sunde, David J. Clarke, Patrick Downey, Tilo Kunath, Mathew H. Horrocks
Abstract
Protein misfolding and aggregation into oligomeric and fibrillar structures is a common feature of many neurogenerative disorders. Single-molecule techniques have enabled characterization of these lowly abundant, highly heterogeneous protein aggregates, previously inaccessible using ensemble averaging techniques. However, they usually rely on the use of recombinantly-expressed labeled protein, or on the addition of amyloid stains that are not protein-specific. To circumvent these challenges, we have made use of a high affinity antibody labeled with orthogonal fluorophores combined with fast-flow microfluidics and single-molecule confocal microscopy to specifically detect α-synuclein, the protein associated with Parkinson's disease. We used this approach to determine the number and size of α-synuclein aggregates down to picomolar concentrations in biologically relevant samples.