Drug Affinity Responsive Target Stability (DARTS) Assay to Detect Interaction Between a Purified Protein and a Small Molecule

Drug affinity responsive target stability (DARTS) assay is used to detect the interaction between a ligand and a protein based on the observation that some ligands can protect the target protein from degradation by proteases when mixed in a solution. To set up the assay, a ligand is first mixed with a purified candidate target protein or a total cell lysate that contains a candidate target protein. Then, different amounts of protease are added to the mixture to allow the enzyme to digest the protein in the mixture. After protease digestion, the candidate target protein is detected by assays such as western blot, silver staining, or Coomassie blue staining. In theory, the candidate protein should be protected by the ligand from protease digestion, which is reflected by higher abundance of the candidate protein in mixtures containing the ligand compared with the control treatment. There are a few significant advantages of DARTS: (a) the ligand does not need to be modified so the native ligand could be used; (b) the candidate target protein could be either purified protein or protein that is present in the total cell lysate; and (c) the assay can be used together with proteomics analysis to identify an unknown target protein. The assay is especially valuable to test the interaction between the ligand and membrane proteins that are often challenging to purify. In this chapter, we use Endosidin2 (ES2) and its target protein Arabidopsis thaliana EXO70A1 (AtEXO70A1) as an example to show the step-by-step procedure of the DARTS assay.