Centre for Targeted Protein Degradation, Division of Biological Chemistry and Drug Discovery, School of Life Sciences, University of Dundee, James Black Centre, Dundee, UK.
Structural Genomics Consortium, University of Toronto, Toronto, ON, Canada.
Methods Mol Biol. 2023;2706:149-165. doi: 10.1007/978-1-0716-3397-7_11.
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.
细胞热转移分析 (CETSA) 基于化合物结合对蛋白质靶标的热稳定性。因此，CETSA 可用于测量化合物的细胞靶标结合和通透性。HiBiT CETSA 方法是定量的，与传统的基于 Western 的 CETSA 相比具有更高的通量。在这里，我们描述了一种利用来自 NanoLuciferase (NanoLuc) 的 HiBiT 标签的 HiBiT CETSA 方案，该标签通过 LgBiT NanoLuc 标签互补产生一个明亮的信号，能够在存在/不存在各种化合物的情况下跟踪温度升高对感兴趣的蛋白质稳定性的影响。HiBiT 标记蛋白暴露于升高的温度会诱导蛋白变性，从而减少 LgBiT 互补和 NanoLuc 信号。由于较高温度下蛋白质的稳定性可能受到化合物结合的影响，因此该方法可用于筛选活细胞或透化细胞中的靶标结合。
