Bellucci Arianna, Fiorentini Chiara, Zaltieri Michela, Missale Cristina, Spano PierFranco
Division of Pharmacology, Department of Molecular and Translational Medicine, University of Brescia, Viale Europa 11, 25123, Brescia, BS, Italy,
Methods Mol Biol. 2014;1174:397-405. doi: 10.1007/978-1-4939-0944-5_27.
The proximity ligation assay (PLA) is a sensitive and specific technique to visualize proteins, their posttranslational modifications and activation state as well as protein-protein interactions. The assay is based on the employment of proximity probes, composed by oligonucleotide-conjugated antibodies, to recognize a couple of specific targets. The binding of probes in close proximity allows for their hybridization by connector oligonucleotides, that can form a circular DNA strand. These DNA circles can then be amplified by polymerase chain reaction. Finally, the conjugation of fluorescence-labelled oligonucleotides with the amplification product allows for the localized detection of individual or interacting proteins in cells and tissues. Here, we describe the use of "in situ" PLA to visualize the localization of protein-protein interactions in intact tissues.
邻近连接分析（PLA）是一种灵敏且特异的技术，可用于可视化蛋白质、其翻译后修饰和激活状态以及蛋白质-蛋白质相互作用。该分析基于使用由寡核苷酸偶联抗体组成的邻近探针来识别一对特定靶点。邻近探针的结合使得它们能够被连接寡核苷酸杂交，后者可形成环状DNA链。这些DNA环随后可通过聚合酶链反应进行扩增。最后，荧光标记的寡核苷酸与扩增产物的结合使得能够在细胞和组织中对单个或相互作用的蛋白质进行定位检测。在此，我们描述了使用“原位”PLA来可视化完整组织中蛋白质-蛋白质相互作用的定位。
