Ligand-macromolecule interactions in live cells by fluorescence correlation spectroscopy.
The receptor concept is the primary theoretical basis for modern pharmacology. Drugs, hormones, neurotransmitters, toxin, and other biologically active substances are referred to as ligands. Ligands exert their actions by way of interaction with receptors/macromolecules. The resulting receptor/macromolecule-ligand complexes produce alterations in physiological processes. Receptor/macromolecule-binding studies most often require the use of radioactively labeled ligands. When the numbers of receptors/macromolecules are few per cell, it is impossible to detect the specific binding because of a high background. Specific interactions between certain ligands and their receptors/macromolecules are, therefore, often overlooked by the conventional binding technique. Fluorescence correlation spectroscopy (FCS) allows detection a ligand-macromolecule interaction in live cells in a tiny confocal volume element (0.2 femtoliter (fL)) at single-molecule detection sensitivity. FCS permits the identification of macromolecules that were not possible to detect before by isotope labeling. The beauty of the FCS technique is that there is no need for separating an unbound ligand from a bound one to calculate the macromolecule bound and free ligand fractions. This study will demonstrate FCS as a sensitive and a rapid technique to study ligand-macromolecule interaction in live cells using fluorescently labeled ligands (Fl-L). This study is of pharmaceutical significance since FCS assay of ligand-macromolecule interactions in live cells is one step forward toward a high throughput drug screening in cell cultures.
Pramanik
Aladdin
A
Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden.
D002470:Cell Survival; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D008024:Ligands; D046911:Macromolecular Substances / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013379:Substrate Specificity
D005456:Fluorescent Dyes; D008024:Ligands; D046911:Macromolecular Substances