Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, 84108, USA.
Biotechniques. 2010 Jul;49(1):519-24. doi: 10.2144/000113452.
Protein interactions are critical for normal biological processes and molecular pathogenesis. While it is important to study these interactions, there are limited assays that are performed inside the cell, in the native cell environment, where the majority of protein-protein interactions take place. Here we present a method of studying protein interactions intracellularly using one protein of interest fused to a localization-controllable enhanced GFP (EGFP) construct and the other protein of interest fused to the red fluorescent protein, DsRed. Nuclear translocation of the EGFP construct is induced by addition of a ligand, and the difference in nuclear localization between the induced and noninduced states of the DsRed construct provides an indication of the interaction between the two proteins. This assay, the nuclear translocation assay (NTA), is introduced here as broadly applicable for studying protein interactions in the native environment inside cells and is demonstrated using forms of the coiled-coil domain from the breakpoint cluster region (Bcr) protein.
蛋白质相互作用对于正常的生物过程和分子发病机制至关重要。虽然研究这些相互作用很重要，但在细胞内、在大多数蛋白质-蛋白质相互作用发生的天然细胞环境中进行的检测有限。在这里，我们提出了一种使用感兴趣的一种蛋白质融合到定位可控的增强型 GFP（EGFP）构建体和另一种蛋白质融合到红色荧光蛋白 DsRed 的方法，在细胞内研究蛋白质相互作用。通过添加配体诱导 EGFP 构建体的核转位，并且 DsRed 构建体的诱导和非诱导状态之间的核定位差异提供了两种蛋白质之间相互作用的指示。该测定法，核转位测定法（NTA），作为在细胞内的天然环境中研究蛋白质相互作用的广泛适用方法被引入，并使用来自断裂点簇区（Bcr）蛋白的卷曲螺旋结构域的形式进行了演示。
