Format

Send to

Choose Destination
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.

Author information

1
BIOSS Centre For Biological Signaling Studies and Department of Molecular Immunology, Biology III, Faculty of Biology, University of Freiburg, Freiburg, Germany.
2
Spemann Graduate School for Biology and Medicine (SGBM), Freiburg, Germany.
3
Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany.

Abstract

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.

PMID:
31825964
PMCID:
PMC6905527
DOI:
10.1371/journal.pbio.3000569
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Public Library of Science Icon for PubMed Central
Loading ...
Support Center