Comprehensive Analysis of Histone-binding Proteins with Multi-Angle Light Scattering

Publication date: Available online 25 January 2020Source: MethodsAuthor(s): Prithwijit Sarkar, Noushin Akhavantabib, Sheena D'ArcyAbstractInteractions 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.
Source: Methods - Category: Molecular Biology Source Type: research