Abstract
Cellular thermal shift assay (CETSA) is based on the thermal stabilization of the protein target by a compound binding. Thus, CETSA can be used to measure a compound’s cellular target engagement and permeability. HiBiT CETSA method is quantitative and has higher throughput compared to the traditional Western-based CETSA. Here, we describe the protocol for a HiBiT CETSA, which utilizes a HiBiT tag derived from the NanoLuciferase (NanoLuc) that upon complementation by LgBiT NanoLuc tag produces a bright signal enabling tracking of the effects of increasing temperature on the stability of a protein-of-interest in the presence/absence of various compounds. Exposure of a HiBiT-tagged protein to increasing temperatures induces protein denaturation and thus decreased LgBiT complementation and NanoLuc signal. As the stability of proteins at higher temperatures can be influenced by the compound binding, this method enables screening for target engagement in living or permeabilized cells.
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Acknowledgements
This research was supported by the Natural Sciences and Engineering Research Council of Canada through a postdoctoral fellowship to V.V. and grant to D.B.L, and by the Structural Genomics Consortium, a registered charity (no: 1097737) that receives funds from Bayer AG, Boehringer Ingelheim, Bristol Myers Squibb, Genentech, Genome Canada through Ontario Genomics Institute [OGI-196], EU/EFPIA/ OICR/McGill/KTH/Diamond Innovative Medicines Initiative 2 Joint Undertaking [EUbOPEN grant 875510], Janssen, Merck KGaA (aka EMD in Canada and US), Pfizer, and Takeda. This project has also received funding from the Innovative Medicines Initiative 2 (IMI2) Joint Undertaking (JU) under grant agreement No 875510. The JU receives support from the European Union’s Horizon 2020 research and innovation program and EFPIA and Ontario Institute for Cancer Research, Royal Institution for the Advancement of Learning McGill University, Kungliga Tekniska Hoegskolan, Diamond Light Source Limited. S.R. is specifically funded by the IMI2 EUbOPEN project. S.H.B was supported by Mitacs Elevate Postdoctoral Fellowship.
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Ramachandran, S., Szewczyk, M., Barghout, S.H., Ciulli, A., Barsyte-Lovejoy, D., Vu, V. (2023). HiBiT Cellular Thermal Shift Assay (HiBiT CETSA). In: Merk, D., Chaikuad, A. (eds) Chemogenomics. Methods in Molecular Biology, vol 2706. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3397-7_11
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DOI: https://doi.org/10.1007/978-1-0716-3397-7_11
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