Jimenez Rosa Meyo, Jeong Joshua, Berger James M
Department of Pharmacology & Molecular Sciences, Johns Hopkins School of Medicine, Baltimore, MD, USA.
Department of Biophysics and Biophysical Chemistry, Johns Hopkins School of Medicine, Baltimore, MD, USA.
Methods Mol Biol. 2025;2928:173-185. doi: 10.1007/978-1-0716-4550-5_14.
Liquid-liquid phase separation (LLPS) is an increasingly studied property of biological macromolecules that can give rise to membrane-less compartments ("condensates") in cells. Analogous to droplets that form after the mixing of oil and water, certain biological macromolecules are also capable of de-mixing from the bulk solution, forming condensates whose contents are partitioned from the rest of the cellular environment. Phase transitions in biology are frequently driven by weak, multivalent protein-protein or protein-nucleic acid interactions, as well as by intrinsically disordered regions (IDRs) of proteins. Fluorescence microscopy is one method commonly used for studying the formation of biological condensates both in vitro and in cellulo. Depending on the system, imaging may be performed with protein alone or with the addition of a nucleic acid ligand or substrate to induce phase separation. Here, we describe a protocol for how to produce dye-labeled samples suitable for studies of in vitro phase separation by a human type II topoisomerase using fluorescence microscopy. The approach does not rely on tags or fusion proteins (which can alter LLPS properties) and is generalizable to any protein with a free, N-terminal primary amine.
液-液相分离（LLPS）是生物大分子一种越来越受关注的特性，它能在细胞中产生无膜区室（“凝聚物”）。类似于油和水混合后形成的液滴，某些生物大分子也能够从整体溶液中分离出来，形成凝聚物，其内含物与细胞环境的其余部分分隔开来。生物学中的相变通常由弱的多价蛋白质-蛋白质或蛋白质-核酸相互作用以及蛋白质的内在无序区域（IDR）驱动。荧光显微镜是体外和细胞内研究生物凝聚物形成常用的一种方法。根据系统的不同，成像可以仅用蛋白质进行，也可以添加核酸配体或底物来诱导相分离。在这里，我们描述了一种通过荧光显微镜制备适用于研究人II型拓扑异构酶体外相分离的染料标记样品的方法。该方法不依赖于标签或融合蛋白（它们会改变液-液相分离特性），并且适用于任何具有游离N端伯胺的蛋白质。
