Abstract：
Proteins drive genome compartmentalization across different length scales. While the identities of these proteins have been well-studied, the physical mechanisms that drive genome organization have remained largely elusive. Studying these mechanisms is challenging owing to a lack of methodologies to parametrize physical models in cellular contexts. Furthermore, because of the complex, entangled, and dense nature of chromatin, conventional live imaging approaches often lack the spatial resolution to dissect these principles. In this chapter, we will describe how to image the interactions of λ-DNA with proteins under purified and cytoplasmic conditions. First, we will outline how to prepare biotinylated DNA, functionalize coverslips with biotin-conjugated poly-ethylene glycol (PEG), and assemble DNA microchannels compatible for the imaging of protein-DNA interactions using total internal fluorescence microscopy. Then we will describe experimental methods to image protein-DNA interactions in vitro and DNA loop extrusion using Xenopus laevis egg extracts.
摘要：
蛋白质驱动不同长度尺度的基因组区隔。虽然这些蛋白质的身份已经得到了充分的研究，驱动基因组组织的物理机制在很大程度上仍然难以捉摸。由于缺乏在细胞环境中参数化物理模型的方法，研究这些机制具有挑战性。此外，因为复杂，纠缠在一起,染色质的浓密性质，传统的实时成像方法通常缺乏空间分辨率来剖析这些原理。在这一章中,我们将描述如何在纯化和细胞质条件下成像λ-DNA与蛋白质的相互作用。首先,我们将概述如何制备生物素化的DNA，用生物素缀合的聚乙二醇（PEG）功能化盖玻片，并使用全内部荧光显微镜组装与蛋白质-DNA相互作用成像兼容的DNA微通道。然后，我们将描述实验方法，以在体外对蛋白质-DNA相互作用进行成像，并使用非洲爪狼卵提取物对DNA环挤出进行成像。
