Laboratory for Genetics of Mitochondrial Disorders, INSERM U1163, Université Paris Descartes-Sorbonne Paris Cité, Institut Imagine, Paris, France.
Methods Mol Biol. 2021;2224:47-60. doi: 10.1007/978-1-0716-1008-4_3.
Like bacterial and cytoplasmic ribosomes, mitoribosomes are large ribonucleoprotein complexes with molecular weights in the range of several million Daltons. Traditionally, studying the assembly of such high molecular weight complexes is done using ultracentrifugation through linear density gradients, which remains the method of choice due to its versatility and superior resolving power in the high molecular weight range. Here, we present a protocol for the analysis of mitoribosomal assembly in heart mitochondrial extracts using linear density sucrose gradients that we have previously employed to characterize the essential role of different mitochondrial proteins in mitoribosomal biogenesis. This protocol details in a stepwise manner a typical mitoribosomal assembly analysis starting with isolation of mitochondria, preparation and ultracentrifugation of the gradients, fractionation and ending with SDS-PAGE, and immunoblotting of the gradient fractions. Even though we provide an example with heart mitochondria, this protocol can be directly applied to virtually all mouse tissues, as well as cultured cells, with little to no modifications.
与细菌和细胞质核糖体一样，线粒体核糖体是分子量在几百万道尔顿范围内的大型核糖核蛋白复合物。传统上，使用通过线性密度梯度的超速离心来研究这种高分子量复合物的组装，由于其多功能性和在高分子量范围内的优越分辨率，它仍然是首选方法。在这里，我们提供了一种使用线性蔗糖密度梯度分析心脏线粒体提取物中线粒体核糖体组装的方案，我们之前曾使用该方案来表征不同线粒体蛋白在线粒体核糖体生物发生中的重要作用。该方案以逐步的方式详细说明了典型的线粒体核糖体组装分析，从线粒体分离开始，制备和超速离心梯度，分级分离，最后进行 SDS-PAGE，并对梯度级分进行免疫印迹。尽管我们提供了一个心脏线粒体的例子，但该方案可以直接应用于实际上所有的小鼠组织以及培养细胞，只需进行很少或无需修改。
