Department of Chemical and Biomolecular Engineering, University of Houston, Houston, TX, USA.
Department of Chemistry, University of Houston, Houston, TX, USA.
Methods Mol Biol. 2023;2551:63-77. doi: 10.1007/978-1-0716-2597-2_6.
We employ time-resolved in situ atomic force microcopy to monitor the growth of individual Aβ40 fibrils and thereby directly measure the fibril growth rates. We describe procedures to express and purify the Aβ peptide and verify its identity, prepare solutions and seeds, quantify the displacements of the growing tips of individual fibrils, and determine their respective growth rates. We discuss approaches to evaluate and minimize the impact of the scanning tip on the monitored processes. We use the distribution of fibril thickness to characterize approximately the fibril structure. The ability to quantify faithfully the growth kinetics of amyloid fibrils empowers exploration of the molecular-level processes of fibril growth that relate to behaviors of amyloid species of laboratory and clinical interest.
我们采用时间分辨的原位原子力显微镜来监测单个 Aβ40 原纤维的生长，从而直接测量原纤维的生长速率。我们描述了表达和纯化 Aβ 肽并验证其身份的程序，准备了溶液和种子，量化了单个原纤维生长尖端的位移，并确定了它们各自的生长速率。我们讨论了评估和最小化扫描尖端对监测过程影响的方法。我们使用原纤维厚度的分布来大致描述原纤维的结构。能够忠实地量化淀粉样原纤维的生长动力学，使我们能够探索与实验室和临床相关的淀粉样物种的行为有关的原纤维生长的分子水平过程。
