Chapter 8
Indirect Detection of Ligand Binding by Thermal Melt
Analysis
Joseph Shaw and Christopher Stubbs
Abstract
A thermal shift assay (TSA) involves measuring the effect of a compound on the thermal stability of a
protein as an indirect measure of ligand binding. In this chapter, we provide a protocol for a conventional
TSA with recombinant/purified proteins using differential scanning fluorimetry (DSF), followed by a
protocol for a Cellular Thermal Shift Assay (CETSA
® ), which measures the soluble cellular protein
remaining after a transient heat shock of live cells to detect intracellular ligand binding.
Key words TSA, Thermal shift assay, Ligand binding, Target engagement, DSF, Differential scanning
fluorimetry, CETSA, Cellular Thermal Shift Assay
1 Introduction
Binding of a ligand to the native, folded state of a protein results in
increased protein stability. This often results in an increase in the
melting temperature of the protein, and it is this effect that is
measured in a thermal shift assay (TSA).
Differential scanning fluorimetry (DSF) is a widely used biophysical technique that can be used to perform a TSA [1–3]. In a
DSF experiment, purified protein (typically recombinant) is mixed
with an environmentally sensitive dye (e.g., SYPRO
® Orange),
whose fluorescence increases with the concentration of unfolded
protein in the mixture. The protein/dye mixture is then subjected
to a temperature ramp in an RT-PCR instrument, where dye fluorescence intensity is recorded as a function of temperature, resulting in a protein melt curve. The midpoint of the melt curve is
defined as the protein melting temperature (T m , Fig. 1a). In a
DSF TSA, the experiment is performed in the absence and presence
of ligands, and a thermal shift ΔT m is calculated (by subtracting the
T m of the protein alone from the T m in the presence of the ligand).
Ligands that induce a ΔT m of !1
C are usually considered as
“hits.”
Tina Daviter et al. (eds.), Protein-Ligand Interactions: Methods and Applications, Methods in Molecular Biology, vol. 2263,
https://doi.org/10.1007/978-1-0716-1197-5_8, © Springer Science+Business Media, LLC, part of Springer Nature 2021
201
Indirect Detection of Ligand Binding by Thermal Melt
Analysis
Joseph Shaw and Christopher Stubbs
Abstract
A thermal shift assay (TSA) involves measuring the effect of a compound on the thermal stability of a
protein as an indirect measure of ligand binding. In this chapter, we provide a protocol for a conventional
TSA with recombinant/purified proteins using differential scanning fluorimetry (DSF), followed by a
protocol for a Cellular Thermal Shift Assay (CETSA
® ), which measures the soluble cellular protein
remaining after a transient heat shock of live cells to detect intracellular ligand binding.
Key words TSA, Thermal shift assay, Ligand binding, Target engagement, DSF, Differential scanning
fluorimetry, CETSA, Cellular Thermal Shift Assay
1 Introduction
Binding of a ligand to the native, folded state of a protein results in
increased protein stability. This often results in an increase in the
melting temperature of the protein, and it is this effect that is
measured in a thermal shift assay (TSA).
Differential scanning fluorimetry (DSF) is a widely used biophysical technique that can be used to perform a TSA [1–3]. In a
DSF experiment, purified protein (typically recombinant) is mixed
with an environmentally sensitive dye (e.g., SYPRO
® Orange),
whose fluorescence increases with the concentration of unfolded
protein in the mixture. The protein/dye mixture is then subjected
to a temperature ramp in an RT-PCR instrument, where dye fluorescence intensity is recorded as a function of temperature, resulting in a protein melt curve. The midpoint of the melt curve is
defined as the protein melting temperature (T m , Fig. 1a). In a
DSF TSA, the experiment is performed in the absence and presence
of ligands, and a thermal shift ΔT m is calculated (by subtracting the
T m of the protein alone from the T m in the presence of the ligand).
Ligands that induce a ΔT m of !1
C are usually considered as
“hits.”
Tina Daviter et al. (eds.), Protein-Ligand Interactions: Methods and Applications, Methods in Molecular Biology, vol. 2263,
https://doi.org/10.1007/978-1-0716-1197-5_8, © Springer Science+Business Media, LLC, part of Springer Nature 2021
201