Brockmöller Sabrina, Molitor Lara Maria, Worek Franz, Rothmiller Simone
Bundeswehr Institute of Pharmacology and Toxicology, 80937 Munich, Bavaria, Germany.
Bio Protoc. 2025 Feb 5;15(3):e5206. doi: 10.21769/BioProtoc.5206.
Different research methods aim to clarify the intracellular trafficking of target proteins or unknown pathways. Currently, existing methods are mostly complex and expensive, requiring expert knowledge. Detailed microscopy for protein co-localization detection or omic technologies, which provide holistic network data, are elaborate, mostly complex, and expensive to apply. Our protocol illustrates a method to track a target protein by detecting expression changes of user-selected marker proteins that directly or indirectly interact with the target. Modulation of protein expression indicates interactions between the target and marker protein. Even without co-localization analysis, the results of the protein expression change are the first insights into the target's fate. Moreover, the use of the cell-sonar is straightforward and affordable, and the results are rapidly available. Furthermore, this method could also be used to determine if and how pathways are affected by compounds added to the cells. In conclusion, our method is adaptable to a wide range of proteins, easy to apply, inexpensive, and expandable with substances that affect proteins. Key features • Easy-to-implement method to track intracellular proteins. • Marker protein expression change demonstrates protein interaction. • Combined data of all marker proteins is used to give an indirect overview of protein localization. • This method is also applicable to different compounds and thus provides information about protein induction or influence on pathways.
不同的研究方法旨在阐明目标蛋白的细胞内运输或未知途径。目前,现有的方法大多复杂且昂贵,需要专业知识。用于蛋白质共定位检测的详细显微镜技术或提供整体网络数据的组学技术都很精细,应用起来大多复杂且昂贵。我们的方案阐述了一种通过检测与目标直接或间接相互作用的用户选择的标记蛋白的表达变化来追踪目标蛋白的方法。蛋白质表达的调节表明目标蛋白与标记蛋白之间的相互作用。即使不进行共定位分析,蛋白质表达变化的结果也是对目标蛋白命运的初步洞察。此外,细胞声呐的使用简单且经济实惠,结果可快速获得。此外,该方法还可用于确定细胞中添加的化合物是否以及如何影响信号通路。总之,我们的方法适用于多种蛋白质,易于应用,成本低廉,并且可通过影响蛋白质的物质进行扩展。关键特性 • 易于实施的细胞内蛋白质追踪方法。 • 标记蛋白表达变化证明蛋白质相互作用。 • 所有标记蛋白的综合数据用于间接概述蛋白质定位。 • 该方法也适用于不同的化合物,从而提供有关蛋白质诱导或对信号通路影响的信息。