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      The cellular thermal shift assay for evaluating drug target interactions in cells.

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          Abstract

          Thermal shift assays are used to study thermal stabilization of proteins upon ligand binding. Such assays have been used extensively on purified proteins in the drug discovery industry and in academia to detect interactions. Recently, we published a proof-of-principle study describing the implementation of thermal shift assays in a cellular format, which we call the cellular thermal shift assay (CETSA). The method allows studies of target engagement of drug candidates in a cellular context, herein exemplified with experimental data on the human kinases p38α and ERK1/2. The assay involves treatment of cells with a compound of interest, heating to denature and precipitate proteins, cell lysis, and the separation of cell debris and aggregates from the soluble protein fraction. Whereas unbound proteins denature and precipitate at elevated temperatures, ligand-bound proteins remain in solution. We describe two procedures for detecting the stabilized protein in the soluble fraction of the samples. One approach involves sample workup and detection using quantitative western blotting, whereas the second is performed directly in solution and relies on the induced proximity of two target-directed antibodies upon binding to soluble protein. The latter protocol has been optimized to allow an increased throughput, as potential applications require large numbers of samples. Both approaches can be completed in a day.

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          Author and article information

          Journal
          Nat Protoc
          Nature protocols
          Springer Science and Business Media LLC
          1750-2799
          1750-2799
          Sep 2014
          : 9
          : 9
          Affiliations
          [1 ] Department of Medical Biochemistry and Biophysics, Division of Biophysics, Karolinska Institutet, Stockholm, Sweden.
          [2 ] Chemical Biology Consortium Sweden, Science for Life Laboratory Stockholm, Division of Translational Medicine and Chemical Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Solna, Sweden.
          Article
          nprot.2014.138
          10.1038/nprot.2014.138
          25101824
          28577ce9-9f81-4b66-adb2-9f277e96f923
          History

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