The cellular thermal shift assay for evaluating drug target interactions in cells
| Autor: | Thomas Lundbäck, Pär Nordlund, Marina Ignatushchenko, Rozbeh Jafari, Hanna Axelsson, Daniel Martinez Molina, Helena Almqvist |
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| Rok vydání: | 2014 |
| Předmět: |
Thermal shift assay
Lysis Hot Temperature biology Kinase Drug discovery MAP Kinase Signaling System Protein Stability Blotting Western Proteins Context (language use) Plasma protein binding Molecular biology General Biochemistry Genetics and Molecular Biology Blot Mitogen-Activated Protein Kinase 14 Drug Delivery Systems Pharmaceutical Preparations Drug Discovery biology.protein Biophysics Humans Antibody Protein Binding |
| Zdroj: | Nature protocols. 9(9) |
| ISSN: | 1750-2799 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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