Abstract
Drug affinity responsive target stability (DARTS) is a relatively quick and straightforward approach to identify potential protein targets for small molecules. It relies on the protection against proteolysis conferred on the target protein by interaction with a small molecule. The greatest advantage of this method is being able to use the native small molecule without having to immobilize or modify it (e.g., by incorporation of biotin, fluorescent, radioisotope, or photoaffinity labels). Here we describe in detail the protocol for performing unbiased DARTS with complex protein lysates to identify binding targets of small molecules and for using DARTS-Western blotting to test, screen, or validate potential small-molecule targets. Although the ideas have mainly been developed from studying molecules in areas of biology that are currently of interest to us and our collaborators, the general principles should be applicable to the analysis of all molecules in nature.
*These authors are contributed equally.
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Acknowledgments
Supported by the US National Institutes of Health grants R01 CA124974 (J.H.), R21 CA149774 (J.H.), U19 AI067769 (W.B.), R01 GM103479 (J.A.L.), R01 GM104610 (J.A.L.), and training grants to M.Y.P. (T32 GM007185) and B.L. (T32 CA009120).
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Pai, M.Y. et al. (2015). Drug Affinity Responsive Target Stability (DARTS) for Small-Molecule Target Identification. In: Hempel, J., Williams, C., Hong, C. (eds) Chemical Biology. Methods in Molecular Biology, vol 1263. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2269-7_22
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DOI: https://doi.org/10.1007/978-1-4939-2269-7_22
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