Single-Molecule Two-Color Coincidence Detection of Unlabeled alpha-Synuclein Aggregates |
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| Authors: | Alexandre Chappard Dr Craig Leighton Dr Rebecca S Saleeb Kiani Jeacock Dr Sarah R Ball Katie Morris Owen Kantelberg Dr Ji-Eun Lee Dr Elsa Zacco Prof Dr Annalisa Pastore Prof Dr Margaret Sunde Dr David J Clarke Dr Patrick Downey Prof Dr Tilo Kunath Dr Mathew H Horrocks |
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| Institution: | 1. EaStCHEM School of Chemistry, The University of Edinburgh, Edinburgh, EH9 3FJ UK;2. School of Medical Sciences, Faculty of Medicine and Health, and Sydney Nano, The University of Sydney, Sydney, NSW 2006 Australia;3. Centre for Human Technologies (CHT), Istituto Italiano di Tecnologia (IIT), Via Enrico Melen, 83, 16152 Genova, Italy;4. European Synchrotron Radiation Facility, 71 Ave des Martyrs, 38000 Grenoble, France;5. UCB Biopharma S.P.R.L., Braine l'Alleud, Belgium;6. Centre for Regenerative Medicine, Institute for Stem Cell Research, School of Biological Sciences, The University of Edinburgh, Edinburgh, EH16 4UU UK |
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| 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. |
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| Keywords: | Aggregation or Oligomerization Fluorescence Microscopy Proteins Single-Molecule |
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