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BMC Res Notes. 2018 Dec 5;11(1):863. doi: 10.1186/s13104-018-3974-5.

Validation of extracellular ligand-receptor interactions by Flow-TriCEPS.

Author information

1
Dualsystems Biotech A.G., Grabenstrasse 11a, 8952, Schlieren, Switzerland.
2
Department of Molecular Mechanisms of Disease, University of Zurich, 8057, Zurich, Switzerland.
3
Molecular Life Science PhD Program, The Life Science Zurich Graduate School, 8057, Zurich, Switzerland.
4
Dualsystems Biotech A.G., Grabenstrasse 11a, 8952, Schlieren, Switzerland. maria.pavlou@dualsystems.com.

Abstract

OBJECTIVE:

The advent of ligand-based receptor capture methodologies, allows the identification of unknown receptor candidates for orphan extracellular ligands. However, further target validation can be tedious, laborious and time-consuming. Here, we present a methodology that provides a fast and cost-efficient alternative for candidate target verification on living cells.

RESULTS:

In the described methodology a ligand of interest (e.g. transferrin, epidermal growth factor or insulin) was conjugated to a linker (TriCEPS) that carries a biotin. To confirm ligand/receptor interactions, the ligand-TriCEPS conjugates were first added onto living cells and cells were subsequently labeled with a streptavidin-fluorophore and analyzed by flow cytometry (thus referred as Flow-TriCEPS). Flow-TriCEPS was also used to validate identified receptor candidates when combined with a siRNA knock down approach (i.e. reduction of expression levels). This approach is versatile as it can be applied for different classes of ligands (proteins, peptides, antibodies) and different cell lines. Moreover, the method is time-efficient since it takes advantage of the large variety of commercially available (and certified) siRNAs.

KEYWORDS:

Candidate verification; Flow-TriCEPS; HATRIC; Knock down; LRC; siRNA

PMID:
30518414
PMCID:
PMC6280402
DOI:
10.1186/s13104-018-3974-5
[Indexed for MEDLINE]
Free PMC Article

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