Belaya Katsiaryna, Tollervey David, Kos Martin
Wellcome Trust Centre for Cell Biology, University of Edinburgh, United Kingdom.
RNA. 2006 May;12(5):921-30. doi: 10.1261/rna.2301806. Epub 2006 Mar 15.
Nucleo-cytoplasmic shuttling is an important feature of proteins involved in nuclear export/import of RNAs, proteins, and also large ribonucleoprotein complexes such as ribosomes. The vast amount of proteomic data available shows that many of these processes are highly dynamic. Therefore, methods are needed to reliably assess whether a protein shuttles between nucleus and cytoplasm, and the kinetics with which it exchanges. Here we describe a combination of the classical heterokaryon assay with fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) techniques, which allows an assessment of the kinetics of protein shuttling in the yeast Saccharomyces cerevisiae.
核质穿梭是参与RNA、蛋白质以及核糖体等大型核糖核蛋白复合物的核输出/输入的蛋白质的一个重要特征。现有的大量蛋白质组学数据表明，其中许多过程具有高度动态性。因此，需要一些方法来可靠地评估一种蛋白质是否在细胞核和细胞质之间穿梭，以及其交换的动力学。在这里，我们描述了经典的异核体测定法与光漂白后荧光恢复（FRAP）和光漂白荧光损失（FLIP）技术的结合，该方法可以评估酿酒酵母中蛋白质穿梭的动力学。
