Drug Affinity Responsive Target Stability (DARTS) Assay to Detect Interaction Between a Purified Protein and a Small Mol
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 s
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Introduction Small molecules are useful and indispensable tools in biology research to understand growth, disease, stress response, etc. [1]. Most of the drugs used in disease control are bioactive small molecules that target endogenous proteins [2, 3]. Small molecules are especially valuable in cell biology research because they allow the manipulation of dynamic cellular processes in a transient, reversible, and dose-dependable manner. For example, multiple bioactive small molecules have been identified to affect plant endomembrane trafficking, and these molecules have been valuable tools
Glenn R. Hicks and Chunhua Zhang (eds.), Plant Chemical Genomics: Methods and Protocols, Methods in Molecular Biology, vol. 2213, https://doi.org/10.1007/978-1-0716-0954-5_15, © Springer Science+Business Media, LLC, part of Springer Nature 2021
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in understanding the mechanisms of dynamic protein transport in plants [4]. The identification of small molecules of interest often starts with chemical library screening. Upon completion of the screening, candidate small molecules need to be further characterized, and the endogenous target protein(s) needs to be identified to better use the small molecule. The process of target identification can be quite challenging and time-consuming. Nowadays, there are various approaches available for the target identification of small molecules [5]. One of the widely used approaches is affinity chromatography combined with mass spectrometry analysis [6]. This approach begins with structure–activity relationship analysis of the small molecule to identify nonessential site so that an affinity tag can be added to the molecule without disrupting the bioactivity of the molecule. A modified small molecule with an affinity tag can be used for affinity chromatography to pull out the endogenous target protein, and the identity of the endogenous target protein can be characterized using mass spectrometry [6]. The most challenging step for affinity chromatography can be to find a way to modify the small molecules without disturbing its activity, and the identified interaction between the small molecule and the candidate target protein often requires further confirmation by other assays [6]. Drug affinity responsive target stability (DARTS) assay contains a few steps [7]. First, the purified protein or cell lysate is mixed with the small molecule of interest. The solvent molecule or the inactive analog molecule will be mixed with the protein as well as necessary controls for the DARTS assay. Then, incubate the mixture of the protein and the small molecule with gentle shaking. The mixture is then aliquoted into small volumes, and the same volume of different concentrations of protease is added to the aliquots of the mixture. After a certain time of protease digestion, stop the digestion reaction by deactivating the protease [7]. The next step is to separate the protease-digested samples on SDS-PAGE and use different approaches to detect the candidate target protein. The protectio
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