Poulard Coralie, Rambaud Juliette, Le Romancer Muriel, Corbo Laura
Cancer Research Center of Lyon, Centre Léon Bérard, UMR INSERM 1052, CNRS 5286, 28, Rue Laënnec, 69373, Lyon Cedex 08, France.
Methods Mol Biol. 2014;1204:135-43. doi: 10.1007/978-1-4939-1346-6_12.
In situ proximity ligation assay (PLA) is a powerful method for detection, localization, and quantification of proteins, protein-protein interactions, and posttranslational modifications. Proteins detected by two specific antibodies are recognized by proximity probes conjugated with complementary oligonucleotides to allow the formation of circular DNA probes when bound in close proximity. Subsequent amplification of this DNA can then be visualized. Here, we describe the in situ PLA method for the detection of the ERα/Src/PI3K complex in breast cancer. We used two different techniques for detecting the signals: fluorescent detection for cell line analysis and bright-field revelation, which is better suited to clinical analysis of patient samples.
原位邻近连接分析（PLA）是一种用于检测、定位和定量蛋白质、蛋白质-蛋白质相互作用以及翻译后修饰的强大方法。由两种特异性抗体检测的蛋白质被与互补寡核苷酸偶联的邻近探针识别，当紧密结合时允许形成环状DNA探针。然后可以对这种DNA的后续扩增进行可视化。在这里，我们描述了用于检测乳腺癌中雌激素受体α/原癌基因酪氨酸蛋白激酶/磷脂酰肌醇-3激酶复合物的原位PLA方法。我们使用了两种不同的技术来检测信号：用于细胞系分析的荧光检测和更适合患者样本临床分析的明场显色。
