Encyclopedia of Experiments: Biological Techniques
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Transcript
The competitive binding assay identifies small molecules that disrupt receptor-ligand interactions by competitively binding to a target receptor, inhibiting natural ligand binding.
To identify a receptor's interactions with its natural ligand in the presence of a receptor-specific small molecule antagonist, begin with a suspension of human T-lymphocyte cells expressing the target receptor.
Add the cell suspension to the wells of a multi-well plate containing increasing antagonist concentrations. Incubate. The antagonist binds to the orthosteric binding site — the receptor's natural ligand-binding site.
Add a fixed concentration of fluorescently-labeled natural ligands to the wells. Incubate.
At low concentrations, the antagonists occupy few orthosteric binding sites, allowing natural ligand binding to the receptors. However, at higher concentrations, the antagonists occupy most of the orthosteric binding sites, allowing minimal natural ligand-receptor binding.
Centrifuge to pellet the cells. Discard the supernatant. Resuspend the cells in paraformaldehyde to fix them, preserving the cellular morphology.
Using a flow cytometer, analyze the fluorescence signals from individual T-lymphocytes expressing the antagonist- and fluorescent ligand-bound receptors.
A dose-dependent decrease in the mean fluorescence intensity with increasing receptor-specific antagonist concentrations indicates competitive binding of the antagonists to the orthosteric receptor-binding sites, inhibiting natural ligand-receptor interaction.
