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.
Highlights
Since the first radioligand binding assay was performed in 19701, the technique has been widely used to characterize the number and specificity of cellular receptors
Ligand-binding assays are fundamental to our understanding of the properties of receptors but existing fluorescence-based binding assays suffer from intra- and intermolecular quenching of the fluorescent ligand
Our results demonstrate that the concentration of fluorescein 5’-isothiocyanate (FITC)-conjugated protein bound to a cell sample can be determined after removing the unbound probe by treating the washed cells with pronase and comparing the fluorescence of the proteolyzed sample to the fluorescence of a known concentration of the free proteolyzed FITC-conjugated protein
Summary
Since the first radioligand binding assay was performed in 19701, the technique has been widely used to characterize the number and specificity of cellular receptors. The desire to generate a cost-effective, risk-averse and environmentally friendly alternative to radioactivity has motivated researchers to develop a ligand-binding assay using fluorescently-labeled ligands rather than radiolabeled ligands[2] Fluorophores such as fluorescein and its derivatives, fluorescein 5’-isothiocyanate (FITC), are among those most commonly conjugated to proteins because they fulfil these criteria and have excellent absorption and emission properties[3]. We developed a “proteolytically-unquenched fluorescence” (PrUF) assay where both the bound FITC-conjugated protein under investigation and a known concentration of the same free FITC-conjugated protein standard are treated with pronase: a mixture of endo- and exo-peptidases with broad-range-specificity[9,10] (Fig. 1) Under these conditions, we demonstrate that the proteolyzed FITC-conjugated proteins display identical fluorescence emission properties to an equimolar amount of FITC conjugated to the simple amine, methylamine (Supplementary Fig. S1). We anticipate that this method will provide researchers with a quantitative, straightforward and convenient alternative to radioligand-binding assays
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