Carvalho Felipe A, Barros Moises R A, Girotto Thierry P F, Perona M Griselda, Oliveira Carla C
Department of Biochemistry, Institute of Chemistry, University of Sao Paulo.
Department of Biochemistry, Institute of Chemistry, University of Sao Paulo;
J Vis Exp. 2020 Nov 9(165). doi: 10.3791/61994.
Pre-mRNA splicing is a very dynamic process that involves many molecular rearrangements of the spliceosome subcomplexes during assembly, RNA processing, and release of the complex components. Glycerol gradient centrifugation has been used for the separation of protein or RNP (RiboNucleoProtein) complexes for functional and structural studies. Here, we describe the utilization of Grafix (Gradient Fixation), which was first developed to purify and stabilize macromolecular complexes for single particle cryo-electron microscopy, to identify interactions between splicing factors that bind transiently to the spliceosome complex. This method is based on the centrifugation of samples into an increasing concentration of a fixation reagent to stabilize complexes. After centrifugation of yeast total extracts loaded on glycerol gradients, recovered fractions are analyzed by dot blot for the identification of the spliceosome sub-complexes and determination of the presence of individual splicing factors.
前体mRNA剪接是一个非常动态的过程，在剪接体亚复合物的组装、RNA加工以及复合物成分释放过程中涉及许多分子重排。甘油梯度离心已被用于分离蛋白质或核糖核蛋白（RNP）复合物，以进行功能和结构研究。在这里，我们描述了Grafix（梯度固定）的应用，该方法最初是为了纯化和稳定用于单颗粒冷冻电子显微镜的大分子复合物而开发的，用于识别与剪接体复合物瞬时结合的剪接因子之间的相互作用。该方法基于将样品离心到浓度不断增加的固定试剂中以稳定复合物。将酵母总提取物加载到甘油梯度上进行离心后，通过点杂交分析回收的组分，以鉴定剪接体亚复合物并确定单个剪接因子的存在。
