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Sci Rep. 2018 Mar 29;8(1):5400. doi: 10.1038/s41598-018-23582-1.

Improved efficiency of in situ protein analysis by proximity ligation using UnFold probes.

Author information

1
Department of Pharmaceutical Biosciences, Pharmaceutical Cell Biology, Uppsala University, Uppsala, Sweden.
2
Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden.
3
Department of Systemic Cell Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany.
4
Department of Pharmaceutical Biosciences, Pharmaceutical Cell Biology, Uppsala University, Uppsala, Sweden. ola.soderberg@farmbio.uu.se.
5
Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden. ulf.landegren@igp.uu.se.

Abstract

We have redesigned probes for in situ proximity ligation assay (PLA), resulting in more efficient localized detection of target proteins. In situ PLA depends on recognition of target proteins by pairs of antibody-oligonucleotide conjugates (PLA probes), which jointly give rise to DNA circles that template localized rolling circle amplification reactions. The requirement for dual recognition of the target proteins improves selectivity by ignoring any cross-reactivity not shared by the antibodies, and it allows detection of protein-protein interactions and post-translational modifications. We herein describe an improved design of the PLA probes -UnFold probes - where all elements required for formation of circular DNA strands are incorporated in the probes. Premature interactions between the UnFold probes are prevented by including an enzymatic "unfolding" step in the detection reactions. This allows DNA circles to form by pairs of reagents only after excess reagents have been removed. We demonstrate the performance of UnFold probes for detection of protein-protein interactions and post-translational modifications in fixed cells and tissues, revealing considerably more efficient signal generation. We also apply the UnFold probes to detect IL-6 in solution phase after capture on solid supports, demonstrating increased sensitivity over both normal sandwich enzyme-linked immunosorbent assays and conventional PLA assays.

PMID:
29599435
PMCID:
PMC5876389
DOI:
10.1038/s41598-018-23582-1
[Indexed for MEDLINE]
Free PMC Article

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