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Author:
Date:
2015-05-20
[Abstract] Protein-protein interaction networks provide a global picture of cellular function and biological processes, and the dysfunction of some interactions causes many diseases, including cancer. The in situ proximity ligation assay (PLA) is a powerful technology capable of detecting the interactions among proteins in fixed tissue and cell samples. The interaction between two proteins is detected using the corresponding two primary antibodies raised in different species. Species-specific secondary antibodies (PLA probes), each with a unique short DNA strand attached to it, bind to the primary antibodies. When the PLA probes are in close proximity (<40 nm), the dna strands can interact through a subsequent addition of two other circle-forming dna oligonucleotides. several-hundredfold replication of the dna circle can occur after the amplification reaction, and a fluorescent signal is generated by labelled complementary oligonucleotide probes. therefore, each detected signal is visualized as an individual fluorescent dot, which can be quantified and assigned to a specific subcellular location based on microscopy images. this revolutionary technique enables us to study the protein complex formation with high specificity and sensitivity compared to the other traditional methods, such as co-immunoprecipitation (co-ip). nm),="" the="" dna="" strands="" can="" interact="" through="" a="" subsequent="" addition="" of="" two="" other="" circle-forming="" dna="" oligonucleotides.="" several-hundredfold="" replication="" of="" the="" dna="" circle="" can="" occur="" after="" the="" amplification="" reaction,="" and="" a="" fluorescent="" signal="" is="" generated="" by="" labelled="" complementary="" oligonucleotide="" probes.="" therefore,="" each="" detected="" signal="" is="" visualized="" as="" an="" individual="" fluorescent="" dot,="" which="" can="" be="" quantified="" and="" assigned="" to="" a="" specific="" subcellular="" location="" based="" on="" microscopy="" images.="" this="" revolutionary="" technique="" enables="" us="" to="" study="" the="" protein="" complex="" formation="" with="" high="" specificity="" and="" sensitivity="" compared="" to="" the="" other="" traditional="" methods,="" such="" as="" co-immunoprecipitation=""> ...
[摘要] 蛋白质 - 蛋白质相互作用网络提供细胞功能和生物过程的全局图像，并且一些相互作用的功能障碍引起许多疾病，包括癌症。原位接近连接测定（PLA）是一种能够检测固定组织和细胞样品中蛋白质之间相互作用的强大技术。使用在不同物种中产生的相应的两种一抗来检测两种蛋白质之间的相互作用。物种特异性二抗（PLA探针），每个具有连接到其的独特的短DNA链，结合一抗。当PLA探针非常接近（<40nm）时，DNA链可以通过随后添加两个其它形成环的DNA寡核苷酸相互作用。 DNA环的几百倍复制可以在扩增反应之后发生，并且通过标记的互补寡核苷酸探针产生荧光信号。因此，每个检测到的信号被可视化为单个荧光点，其可以基于显微镜图像被量化并分配到特定的亚细胞位置。这种革命性的技术使我们能够与其他传统方法，如共免疫沉淀（Co-IP）相比，以高特异性和灵敏度研究蛋白质复合物形成。
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