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. 2023:2706:149-165.
doi: 10.1007/978-1-0716-3397-7_11.

HiBiT Cellular Thermal Shift Assay (HiBiT CETSA)

Sarath Ramachandran  1 , Magdalena Szewczyk  2 , Samir H Barghout  2   3 , Alessio Ciulli  1 , Dalia Barsyte-Lovejoy  2   3 , Victoria Vu  4
Affiliations

HiBiT Cellular Thermal Shift Assay (HiBiT CETSA)

Sarath Ramachandran et al. Methods Mol Biol. 2023.

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.

Keywords: CETSA; Drug discovery; HiBiT; Split nanoluciferase; Target engagement; Thermal shift.

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