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PLoS Biol. 2019 Dec 11;17(12):e3000569. doi: 10.1371/journal.pbio.3000569. eCollection 2019 Dec.

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

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BIOSS Centre For Biological Signaling Studies and Department of Molecular Immunology, Biology III, Faculty of Biology, University of Freiburg, Freiburg, Germany.
Spemann Graduate School for Biology and Medicine (SGBM), Freiburg, Germany.
Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany.


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.

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