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Sci Rep. 2016 May 10;6:25769. doi: 10.1038/srep25769.

Development of a quantitative fluorescence-based ligand-binding assay.

Author information

1
Department of Biology, Maynooth University, Maynooth, Co. Kildare, Ireland.
2
School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin 2, Ireland.

Abstract

A major goal of biology is to develop a quantitative ligand-binding assay that does not involve the use of radioactivity. Existing fluorescence-based assays have a serious drawback due to fluorescence quenching that accompanies the binding of fluorescently-labeled ligands to their receptors. This limitation of existing fluorescence-based assays prevents the number of cellular receptors under investigation from being accurately measured. We have developed a method where FITC-labeled proteins bound to a cell surface are proteolyzed extensively to eliminate fluorescence quenching and then the fluorescence of the resulting sample is compared to that of a known concentration of the proteolyzed FITC-protein employed. This step enables the number of cellular receptors to be measured quantitatively. We expect that this method will provide researchers with a viable alternative to the use of radioactivity in ligand binding assays.

PMID:
27161290
PMCID:
PMC4861924
DOI:
10.1038/srep25769
[Indexed for MEDLINE]
Free PMC Article

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