期刊
PLOS BIOLOGY
卷 17, 期 12, 页码 -出版社
PUBLIC LIBRARY SCIENCE
DOI: 10.1371/journal.pbio.3000569
关键词
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资金
- Deutsche Forschungsgemeinschaft [TRR130-P02]
- Max Planck Society
- Excellence Initiative of the German Federal Government [EXC294]
- Excellence Initiative of the German State Government [EXC294]
- Spemann Graduate School for Biology and Medicine (SGBM)
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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