Blancas-Mejia Luis M, Misra Pinaki, Dick Christopher J, Marin-Argany Marta, Redhage Keely R, Cooper Shawna A, Ramirez-Alvarado Marina
Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, USA.
Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain.
Methods Mol Biol. 2019;1873:123-153. doi: 10.1007/978-1-4939-8820-4_8.
Common biophysical techniques like absorption and fluorescence spectroscopy, microscopy, and light scattering studies have been in use to investigate fibril assembly for a long time. However, there is sometimes a lack of consensus from the findings of an individual technique when compared in parallel with the other techniques. In this chapter, we aim to provide a concise compilation of techniques that can effectively be used to obtain a comprehensive representation of the structural, aggregation, and toxicity determinants in immunoglobulin light chain amyloidosis. We start by giving a brief introduction on amyloid assembly and the advantages of using simple and readily available techniques to study aggregation. After an overview on preparation of protein to set up parallel experiments, we provide a systematic description of the in vitro techniques used to study aggregation in AL protein. Additionally, we thoroughly discuss the steps needed in our experience during the individual experiments for better reproducibility and data analysis.
诸如吸收光谱和荧光光谱、显微镜技术以及光散射研究等常见的生物物理技术，长期以来一直被用于研究纤维组装。然而，与其他技术并行比较时，个别技术的研究结果有时缺乏一致性。在本章中，我们旨在简要汇编一些技术，这些技术可有效用于全面呈现免疫球蛋白轻链淀粉样变性中的结构、聚集和毒性决定因素。我们首先简要介绍淀粉样蛋白组装以及使用简单且易于获得的技术研究聚集的优势。在概述用于开展平行实验的蛋白质制备方法后，我们系统描述用于研究AL蛋白聚集的体外技术。此外，我们还深入讨论了根据我们的经验在各个实验中为实现更好的可重复性和数据分析所需的步骤。
