PDF(Pubmed)
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.
Pathological protein aggregates in neurodegenerative disorders are difficult to characterise using current methods. We present a novel single‐molecule detection method to specifically detect and characterise α‐synuclein aggregates at picomolar concentrations. We demonstrate the ability to detect aggregates in biologically relevant samples.
Pathological protein aggregates in neurodegenerative disorders are difficult to characterise using current methods. We present a novel single‐molecule detection method to specifically detect and characterise α‐synuclein aggregates at picomolar concentrations. We demonstrate the ability to detect aggregates in biologically relevant samples.
摘要：
蛋白质错误折叠和聚集成寡聚和纤维结构是许多神经发生障碍的共同特征。单分子技术已经能够表征这些低丰度，高度异质的蛋白质聚集体，以前无法使用集合平均技术访问。然而,它们通常依赖于使用重组表达的标记蛋白，或添加非蛋白质特异性的淀粉样蛋白染色。为了规避这些挑战，我们已经利用正交荧光团标记的高亲和力抗体，结合快速流动微流体和单分子共聚焦显微镜来特异性检测α-突触核蛋白，与帕金森病相关的蛋白质。我们使用这种方法来确定生物相关样品中降至皮摩尔浓度的α-突触核蛋白聚集体的数量和大小。
神经退行性疾病中的病理蛋白聚集体难以用现有方法表征。我们提出了一种新颖的单分子检测方法，可以在皮摩尔浓度下特异性检测和表征α突触核蛋白聚集体。我们证明了在生物相关样品中检测聚集体的能力。
神经退行性疾病中的病理蛋白聚集体难以用现有方法表征。我们提出了一种新颖的单分子检测方法，可以在皮摩尔浓度下特异性检测和表征α突触核蛋白聚集体。我们证明了在生物相关样品中检测聚集体的能力。
