Maszczak-Seneczko Dorota, Sosicka Paulina, Olczak Teresa, Olczak Mariusz
Laboratory of Biochemistry, Faculty of Biotechnology, University of Wroclaw, Joliot-Curie 14A, 50-383, Wrocław, Poland.
Methods Mol Biol. 2016;1496:133-43. doi: 10.1007/978-1-4939-6463-5_11.
In situ proximity ligation assay (PLA) is a novel, revolutionary technique that can be employed to visualize protein complexes in fixed cells and tissues. This approach enables demonstration of close (i.e., up to 40 nm) proximity between any two proteins of interest that can be detected using a pair of specific antibodies that have been raised in distinct species. Primary antibodies bound to the target proteins are subsequently recognized by two PLA probes, i.e., secondary antibodies conjugated with oligonucleotides that anneal when brought into close proximity in the presence of two connector oligonucleotides and a DNA ligase forming a circular DNA molecule. In the next step, the resulting circular DNA is amplified by a rolling circle polymerase. Finally, fluorescent oligonucleotide probes hybridize to complementary fragments of the amplified DNA molecule, forming a typical, spot-like pattern of PLA signal that reflects subcellular localization of protein complexes. Here we describe the use of in situ PLA in adherent cultures of mammalian cells in order to visualize interactions between Golgi-resident, functionally related glycosyltransferases and nucleotide sugar transporters relevant to N-glycan biosynthesis.
原位邻近连接分析（PLA）是一种新颖的革命性技术，可用于在固定细胞和组织中可视化蛋白质复合物。这种方法能够证明任何两种感兴趣的蛋白质之间的紧密（即高达40纳米）邻近性，这可以使用在不同物种中产生的一对特异性抗体来检测。与靶蛋白结合的一抗随后被两种PLA探针识别，即与寡核苷酸缀合的二抗，当在两种连接子寡核苷酸和DNA连接酶存在下紧密接近时，它们会退火形成环状DNA分子。在下一步中，产生的环状DNA通过滚环聚合酶进行扩增。最后，荧光寡核苷酸探针与扩增的DNA分子的互补片段杂交，形成典型的点状PLA信号模式，反映蛋白质复合物的亚细胞定位。在这里，我们描述了原位PLA在哺乳动物细胞贴壁培养中的应用，以便可视化与N-聚糖生物合成相关的高尔基体驻留、功能相关的糖基转移酶和核苷酸糖转运蛋白之间的相互作用。
