A new branched proximity hybridization assay for the quantification of nanoscale protein-protein proximity.,PLOS Biology

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A new branched proximity hybridization assay for the quantification of nanoscale protein-protein proximity.
PLOS Biology ( IF 7.8 ) Pub Date : 2019-12-11 , DOI: 10.1371/journal.pbio.3000569
Shuangshuang Zheng ₁ , Melanie Sieder _{1,

2} , Michael Mitterer ₃ , Michael Reth _{1,

3} , Marco Cavallari _{1,

3} , Jianying Yang _{1,

3}

Affiliation

Membrane proteins are organized in nanoscale compartments. Their reorganization plays a crucial role in receptor activation and cell signaling. To monitor the organization and reorganization of membrane proteins, we developed a new branched proximity hybridization assay (bPHA) allowing better quantification of the nanoscale protein-protein proximity. In this assay, oligo-coupled binding probes, such as aptamer, nanobody, and antibodies, are used to translate the proximity of target proteins to the proximity of oligos. The closely positioned oligos then serve as a template for a maximum of 400-fold branched DNA (bDNA) signal amplification. The amplified bPHA signal is recorded by flow cytometer, thus enabling proximity studies with high throughput, multiplexing, and single-cell resolution. To demonstrate the potential of the bPHA method, we measured the reorganization of the immunoglobulin M (IgM)- and immunoglobulin D (IgD)-class B cell antigen receptor (BCR) on the plasma membrane and the recruitment of spleen tyrosine kinase (Syk) to the BCR upon B lymphocyte activation.

中文翻译：

一种新的分支邻近杂交测定法，用于量化纳米级蛋白质-蛋白质邻近度。

膜蛋白组织在纳米级隔室中。它们的重组在受体激活和细胞信号传导中起着至关重要的作用。为了监测膜蛋白的组织和重组，我们开发了一种新的分支邻近杂交测定法（bPHA），可以更好地量化纳米级蛋白质与蛋白质的接近度。在该测定中，寡核苷酸偶联的结合探针，例如适体，纳米抗体和抗体，用于将靶蛋白的邻近翻译为寡核苷酸。然后，位置紧密的寡核苷酸可作为模板进行最大400倍分支DNA（bDNA）信号扩增。扩增的bPHA信号通过流式细胞仪记录，因此能够以高通量，多路复用和单细胞分辨率进行邻近研究。为了证明bPHA方法的潜力，

更新日期：2019-12-11

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