title: Development of a quantitative fluorescence-based ligand-binding assay.
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.
authors.affiliation: Department of Biology, Maynooth University, Maynooth, Co. Kildare, Ireland.|School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin 2, Ireland.
authors.forename: Conor J
authors.initials: CJ
authors.lastname: Breen
authors.affiliation: School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin 2, Ireland.
authors.forename: Mathilde
authors.initials: M
authors.lastname: Raverdeau
authors.affiliation: School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin 2, Ireland.
authors.forename: H Paul
authors.initials: HP
authors.lastname: Voorheis
mesh_terms: D000818:Animals; D001681:Biological Assay / Q000379:methods*; D002417:Cattle; D005407:Flagella / Q000378:metabolism; D019793:Fluorescein / Q000378:metabolism; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism; D005453:Fluorescence; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D008744:Methylamines; D011402:Pronase / Q000378:metabolism; D059748:Proteolysis; D011522:Protons; D012710:Serum Albumin, Bovine / Q000378:metabolism; D014168:Transferrin / Q000378:metabolism; D014346:Trypanosoma brucei brucei / Q000378:metabolism
chemical_list: D008024:Ligands; D008744:Methylamines; D011522:Protons; D014168:Transferrin; D012710:Serum Albumin, Bovine; C027451:methylamine; D011402:Pronase; D016650:Fluorescein-5-isothiocyanate; D019793:Fluorescein