HiBiT Cellular Thermal Shift Assay (HiBiT CETSA)

Sarath Ramachandran, Magdalena Szewczyk, Samir H. Barghout, Alessio Ciulli, Dalia Barsyte-Lovejoy, Victoria Vu (Lead / Corresponding author)

Research output: Chapter in Book/Report/Conference proceedingChapter (peer-reviewed)peer-review

3 Citations (Scopus)
45 Downloads (Pure)

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 languageEnglish
Title of host publicationChemogenomics
Subtitle of host publicationMethods and Protocols
EditorsDaniel Merk, Apirat Chaikuad
Place of PublicationNew York
PublisherHumana Press
Pages149-165
Number of pages17
Edition1
ISBN (Electronic)9781071633977
ISBN (Print)9781071633960 (hbk), 9781071633991 (pbk)
DOIs
Publication statusPublished - 2023

Publication series

NameMethods in Molecular Biology
Volume2706
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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