Mendez Roberto, Banerjee Santanu
Department of Surgery, Miller School of Medicine, University of Miami, Miami, FL, USA.
Bio Protoc. 2017 Nov 5;7(21). doi: 10.21769/BioProtoc.2602.
In this protocol, we describe proximal ligation assay (PLA), an antibody-based detection method for protein-protein interaction. This method relies on specific binding of individual primary antibodies to the two putative interacting proteins. The primary antibodies need to have different hosts. The secondary antibodies against the two hosts have complementary oligonucleotide moieties attached to them. If the two antigens are in close proximity (presumably interacting with each other), the complementary oligonucleotides can anneal and fluorescent nucleotides can be incorporated in a single DNA polymerization step. Under a microscope, these reactions appear as punctate fluorescent spots, indicating successful PLA reaction and suggesting protein-protein interaction between the two antigens.
在本方案中，我们描述了近端连接分析（PLA），这是一种基于抗体的蛋白质-蛋白质相互作用检测方法。该方法依赖于单个一抗与两种假定相互作用的蛋白质的特异性结合。一抗需要具有不同的宿主。针对这两种宿主的二抗连接有互补的寡核苷酸部分。如果两种抗原靠得很近（推测它们相互作用），互补寡核苷酸可以退火，并且荧光核苷酸可以在单个DNA聚合步骤中掺入。在显微镜下，这些反应表现为点状荧光斑点，表明PLA反应成功，并提示两种抗原之间存在蛋白质-蛋白质相互作用。
