title: Drug affinity responsive target stability (DARTS) for small-molecule target identification.
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.
authors.affiliation: Molecular Biology Institute, University of California Los Angeles, Los Angeles, CA, USA.
authors.forename: Melody Y
authors.initials: MY
authors.lastname: Pai
authors.forename: Brett
authors.initials: B
authors.lastname: Lomenick
authors.forename: Heejun
authors.initials: H
authors.lastname: Hwang
authors.forename: Robert
authors.initials: R
authors.lastname: Schiestl
authors.forename: William
authors.initials: W
authors.lastname: McBride
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Loo
authors.forename: Jing
authors.initials: J
authors.lastname: Huang
mesh_terms: D008024:Ligands; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D054852:Small Molecule Libraries
chemical_list: D008024:Ligands; D011506:Proteins; D054852:Small Molecule Libraries