Interactions between histones and their binding partners are an important aspect of chromatin biology. Determining the stoichiometry of histone-containing complexes is an important pre-requisite for performing in vitro biochemical, biophysical and structural analyses. In this article, we detail how Size Exclusion Chromatography (SEC) coupled to Multi-Angle Light Scattering (MALS) can be used to study histone chaperones and their complexes. Our protocol details system setup, sample preparation, data collection, and data interpretation. We provide tips on designing an informative SEC-MALS experiment, using histone chaperones Nap1 and Vps75 as demonstrative examples. We outline recommendations to overcome specific challenges such as protein oligomerization, heterogeneity, and non-specific binding. We find SEC-MALS to be a robust and user-friendly approach for characterizing histone-binding proteins and their complexes.

中文翻译：

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多角度光散射综合分析组蛋白结合蛋白


组蛋白及其结合伴侣之间的相互作用是染色质生物学的一个重要方面。确定含组蛋白复合物的化学计量是进行体外生化、生物物理和结构分析的重要先决条件。在本文中，我们详细介绍了如何使用尺寸排阻色谱 (SEC) 与多角度光散射 (MALS) 联用来研究组蛋白伴侣及其复合物。我们的协议详细介绍了系统设置、样品制备、数据收集和数据解释。我们使用组蛋白伴侣 Nap1 和 Vps75 作为示范性示例，提供设计信息丰富的 SEC-MALS 实验的技巧。我们概述了克服蛋白质寡聚化、异质性和非特异性结合等特定挑战的建议。我们发现 SEC-MALS 是一种强大且用户友好的方法，用于表征组蛋白结合蛋白及其复合物。