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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.
Original language | English |
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Title of host publication | Chemogenomics |
Subtitle of host publication | Methods and Protocols |
Editors | Daniel Merk, Apirat Chaikuad |
Place of Publication | New York |
Publisher | Humana Press |
Pages | 149-165 |
Number of pages | 17 |
Edition | 1 |
ISBN (Electronic) | 9781071633977 |
ISBN (Print) | 9781071633960 (hbk), 9781071633991 (pbk) |
DOIs | |
Publication status | Published - 2023 |
Publication series
Name | Methods in Molecular Biology |
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Volume | 2706 |
ISSN (Print) | 1064-3745 |
ISSN (Electronic) | 1940-6029 |
Keywords
- Proteins
- Temperature
- Hot Temperature
- Druggable human proteome
- Annotated chemogenomic compounds
- Drug targets
- Target validation
- Targeted chemical libraries
- Novel drugs
- Split nanoluciferase
- HiBiT
- CETSA
- Thermal shift
- Target engagement
- Drug discovery
ASJC Scopus subject areas
- Genetics
- Molecular Biology
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EUbOPEN: Enabling and Unlocking biology in the OPEN (Joint with Goethe University Frankfurt, University of Oxford, Karolinska Institute and 9 others)
Ciulli, A. (Investigator)
COMMISSION OF THE EUROPEAN COMMUNITIES
1/05/20 → 30/04/25
Project: Research