Koos Björn, Söderberg Ola
Max Planck Institute for Molecular Physiology, Department of Systemic Cell Biology, Dortmund, Germany.
Department of Immunology, Genetics and Pathology, Uppsala University, Science for Life Laboratory, Uppsala, Sweden.
Curr Protoc Protein Sci. 2016 Aug 1;85:19.28.1-19.28.13. doi: 10.1002/cpps.9.
Proximity-dependent hybridization chain reaction (proxHCR) is a novel technique for detection of protein interaction, post-translational modifications (PTMs), or protein expression. The method is based upon antibodies targeting the proteins of interest that are covalently conjugated to DNA oligonucleotides, which enables the induction of a hybridization chain reaction (HCR) to generate a fluorescent signal visible under a microscope. In contrast to the in situ proximity ligation assay (in situ PLA), which is another method that utilizes antibody-DNA conjugates to detect protein interactions, proxHCR does not require enzymatic steps. This makes proxHCR an inexpensive alternative to in situ PLA. Another potential advantage might be that proxHCR could more readily be adapted for use in automated staining procedures and in point-of-care devices, as all reagents can be stored at room temperature. This unit describes how the oligonucleotide system for proxHCR can be designed and a protocol for how to perform proxHCR is presented. © 2016 by John Wiley & Sons, Inc.
邻近依赖杂交链式反应（proxHCR）是一种用于检测蛋白质相互作用、翻译后修饰（PTM）或蛋白质表达的新技术。该方法基于靶向目标蛋白质的抗体，这些抗体与DNA寡核苷酸共价结合，从而能够诱导杂交链式反应（HCR）产生在显微镜下可见的荧光信号。与另一种利用抗体 - DNA偶联物检测蛋白质相互作用的原位邻近连接分析（原位PLA）不同，proxHCR不需要酶促步骤。这使得proxHCR成为原位PLA的一种低成本替代方法。另一个潜在优势可能是，由于所有试剂都可以在室温下储存，proxHCR可以更容易地应用于自动化染色程序和即时检测设备。本单元描述了如何设计用于proxHCR的寡核苷酸系统，并给出了进行proxHCR的实验方案。© 2016约翰威立父子公司版权所有
