Indirect Detection of Ligand Binding by Thermal Melt Analysis

Area of Science:

• Biochemistry
• Molecular Biology
• Pharmacology

Background:

• Thermal Shift Assay (TSA) is a biophysical technique.
• It indirectly measures ligand-protein interactions by assessing changes in protein thermal stability.
• Detecting intracellular ligand binding requires cellular-based assays.

Purpose of the Study:

• To provide protocols for performing both conventional TSA and Cellular Thermal Shift Assay (CETSA®).
• To enable the detection of ligand binding to purified proteins and intracellular targets.
• To offer a comprehensive guide for researchers utilizing thermal shift assays.

Main Methods:

• Conventional TSA using differential scanning fluorimetry (DSF) on recombinant/purified proteins.
• Cellular Thermal Shift Assay (CETSA®) involving heat shock of live cells.
• Quantification of soluble cellular protein remaining after heat treatment to infer target engagement.

Main Results:

• Established protocols for implementing TSA and CETSA®.
• Demonstrated the utility of DSF for purified protein thermal stability.
• Validated CETSA® for detecting intracellular ligand binding through soluble protein measurement.

Conclusions:

• TSA and CETSA® are versatile methods for studying ligand-protein interactions.
• CETSA® provides a powerful approach for assessing drug target engagement within live cells.
• These protocols facilitate the study of molecular interactions in both purified and cellular systems.