Abstract
We introduce three assays for analyzing ligand-receptor interactions based on the specific conjugation of ligands to SNAP-tag fusion proteins. Conjugation of ligands to different SNAP-tag fusions permits the validation of suspected interactions in cell extracts and fixed cells as well as the establishment of high-throughput assays. The different assays allow the analysis of strong and weak interactions. Conversion of ligands into SNAP-tag substrates thus provides access to a powerful toolbox for the analysis of their interactions with proteins.
Highlights
Methods for the detection of ligand-receptor interactions are a crucial part of drug discovery and chemical biology in general [1,2,3,4,5]
SNAP-based timeresolved fluorescence resonance energy transfer (TR-FRET) Assay To develop a method for the quantification of ligand-receptor interactions that is suitable for high-throughput applications, we took advantage of both SNAP-tag and TR-FRET technology using lanthanides (Figure 1B)
In the SNAP-tag-based TR-FRET assay, as shown in Figure 2A, a ligand was conjugated via SNAP-tag to EGFP acting as FRET acceptor
Summary
Methods for the detection of ligand-receptor interactions are a crucial part of drug discovery and chemical biology in general [1,2,3,4,5]. For the identification of the protein targets of a given ligand (i.e. a drug or bioactive small molecule), affinity chromatography is most commonly used [6]. For a detailed biophysical characterization of a known ligand-receptor interaction, approaches such as isothermal titration calorimetry, surface plasmon resonance, NMR or X-ray crystallography are chosen [4]. Additional factors such as availability, purity, solubility, and stability of the protein of interest influence the assay choice. The development of suitable methods for the detection of ligand-receptor interactions can still be a formidable challenge and the availability of tools to rapidly establish a variety of complementary assay systems would help to overcome this challenge
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