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

Nature Protocols volume 9, pages 21002122 (2014) | Download Citation


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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D.M.M., R.J., M.I. and P.N. acknowledge Karolinska Institutet (Distinguished Professor Award), the Swedish Research Council (Vetenskapsrådet) and the Swedish Cancer Society (Cancerfonden). T.L., H. Almqvist and H. Axelsson acknowledge Karolinska Institutet and the Swedish Research Council, which funds Chemical Biology Consortium Sweden, a national infrastructure for research in the field of chemical Biology, as well as Biovitrum for a generous donation of instruments and compound libraries when Chemical Biology Consortium Sweden was inaugurated. We are also grateful to C. Zaniol at PerkinElmer for great support in the development of the AlphaScreen assays.

Author information


  1. Department of Medical Biochemistry and Biophysics, Division of Biophysics, Karolinska Institutet, Stockholm, Sweden.

    • Rozbeh Jafari
    • , Marina Ignatushchenko
    • , Pär Nordlund
    •  & Daniel Martinez Molina
  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.

    • Helena Almqvist
    • , Hanna Axelsson
    •  & Thomas Lundbäck


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R.J., H. Axelsson, H. Almqvist, M.I., T.L., P.N. and D.M.M. conceived the study; R.J., H. Axelsson, H. Almqvist, T.L. and D.M.M. designed the experiments and the high-throughput formatting of the CETSA procedure; western blot and the AlphaScreen experiments were conducted by R.J., D.M.M., H. Axelsson, H. Almqvist and T.L.; and the manuscript was prepared by R.J., H. Axelsson, H. Almqvist, M.I., T.L., P.N. and D.M.M.

Competing interests

D.M.M. and P.N. are the founders of Pelago Bioscience AB. The remaining authors declare no competing financial interests.

Corresponding authors

Correspondence to Thomas Lundbäck or Pär Nordlund or Daniel Martinez Molina.

Integrated supplementary information

Supplementary information

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  1. 1.

    Supplementary Figure 1

    Cellular integrity after heating.

  2. 2.

    Supplementary Figure 2

    Chemical structures.

  3. 3.

    Supplementary Figure 3

    Negative control compound on p38α.

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    Supplementary Figure 4

    ITDRFCETSA confirmation of CBK derived hit compounds.

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    Supplementary Data

    Positive control compound on ERK1/2.

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