Porter Elizabeth G, Connelly Katelyn E, Dykhuizen Emily C
Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University.
Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University;
J Vis Exp. 2017 Oct 2(128):55369. doi: 10.3791/55369.
Elucidation of the binding properties of chromatin-targeting proteins can be very challenging due to the complex nature of chromatin and the heterogeneous nature of most mammalian chromatin-modifying complexes. In order to overcome these hurdles, we have adapted a sequential salt extraction (SSE) assay for evaluating the relative binding affinities of chromatin-bound complexes. This easy and straightforward assay can be used by non-experts to evaluate the relative difference in binding affinity of two related complexes, the changes in affinity of a complex when a subunit is lost or an individual domain is inactivated, and the change in binding affinity after alterations to the chromatin landscape. By sequentially re-suspending bulk chromatin in increasing amounts of salt, we are able to profile the elution of a particular protein from chromatin. Using these profiles, we are able to determine how alterations in a chromatin-modifying complex or alterations to the chromatin environment affect binding interactions. Coupling SSE with other in vitro and in vivo assays, we can determine the roles of individual domains and proteins on the functionality of a complex in a variety of chromatin environments.
由于染色质的复杂性质以及大多数哺乳动物染色质修饰复合物的异质性，阐明靶向染色质的蛋白质的结合特性可能极具挑战性。为了克服这些障碍，我们采用了一种连续盐提取（SSE）测定法来评估染色质结合复合物的相对结合亲和力。这种简单直接的测定法非专业人员也可使用，用于评估两种相关复合物结合亲和力的相对差异、亚基缺失或单个结构域失活时复合物亲和力的变化，以及染色质格局改变后结合亲和力的变化。通过在不断增加的盐量中依次重悬大量染色质，我们能够分析特定蛋白质从染色质上的洗脱情况。利用这些洗脱曲线，我们能够确定染色质修饰复合物的改变或染色质环境的改变如何影响结合相互作用。将SSE与其他体外和体内测定法相结合，我们可以确定单个结构域和蛋白质在各种染色质环境中对复合物功能的作用。
