Chapter 6
Measuring the K D of Protein–Ligand Interactions Using
Microscale Thermophoresis
Shih-Chia Tso and Chad A. Brautigam
Abstract
Microscale thermophoresis (MST) has become a widely used technique to determine the K D or EC 50 of
protein–ligand interactions. The method exploits the tendency of macromolecules to migrate along a
thermal gradient (i.e., thermophoresis). Differences in thermophoresis as a function of the liganded state
of a macromolecule can be measured and assembled into a binding curve that can be analyzed to yield K D .
In this protocol, we outline a simple experiment designed for new MST users, with the goal of using readily
available, inexpensive materials to plan, execute, and analyze an MST experiment.
Key words Microscale thermophoresis, Protein–ligand interactions, Protein–protein interactions,
K D , Affinity measurement
1 Introduction
Since it became available in a commercially distributed instrument,
microscale thermophoresis (MST) has enjoyed rapid growth and
widespread adoption. Although many kinds of experiments can be
conducted in an MST instrument [1], most MST studies aim to
characterize the interaction(s) between a fluorescently labeled
receptor (termed “B*” herein) and an unlabeled ligand (called
“A” henceforth; the 1:1 complex between the two is “AB*”). In
such studies, a mixture of the receptor and ligand is placed into a
glass capillary tube (Fig. 1a). A portion of the tube near its center is
illuminated with visible light that stimulates the fluorophore associated with the receptor. Fluorescence emitted from the capillary is
monitored via a detector configured as a confocal microscope.
Next, an infrared (IR) laser is actuated, rapidly forming a temperature gradient at the monitored part of the capillary. Through a
process called “thermophoresis,” the labeled receptor molecules
display a net movement along this gradient, changing the net
fluorescence recorded (Fig. 1b). Thermophoresis is usually “positive”; i.e., the molecules move from hotter to colder areas of the
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_6, © Springer Science+Business Media, LLC, part of Springer Nature 2021
161
Measuring the K D of Protein–Ligand Interactions Using
Microscale Thermophoresis
Shih-Chia Tso and Chad A. Brautigam
Abstract
Microscale thermophoresis (MST) has become a widely used technique to determine the K D or EC 50 of
protein–ligand interactions. The method exploits the tendency of macromolecules to migrate along a
thermal gradient (i.e., thermophoresis). Differences in thermophoresis as a function of the liganded state
of a macromolecule can be measured and assembled into a binding curve that can be analyzed to yield K D .
In this protocol, we outline a simple experiment designed for new MST users, with the goal of using readily
available, inexpensive materials to plan, execute, and analyze an MST experiment.
Key words Microscale thermophoresis, Protein–ligand interactions, Protein–protein interactions,
K D , Affinity measurement
1 Introduction
Since it became available in a commercially distributed instrument,
microscale thermophoresis (MST) has enjoyed rapid growth and
widespread adoption. Although many kinds of experiments can be
conducted in an MST instrument [1], most MST studies aim to
characterize the interaction(s) between a fluorescently labeled
receptor (termed “B*” herein) and an unlabeled ligand (called
“A” henceforth; the 1:1 complex between the two is “AB*”). In
such studies, a mixture of the receptor and ligand is placed into a
glass capillary tube (Fig. 1a). A portion of the tube near its center is
illuminated with visible light that stimulates the fluorophore associated with the receptor. Fluorescence emitted from the capillary is
monitored via a detector configured as a confocal microscope.
Next, an infrared (IR) laser is actuated, rapidly forming a temperature gradient at the monitored part of the capillary. Through a
process called “thermophoresis,” the labeled receptor molecules
display a net movement along this gradient, changing the net
fluorescence recorded (Fig. 1b). Thermophoresis is usually “positive”; i.e., the molecules move from hotter to colder areas of the
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_6, © Springer Science+Business Media, LLC, part of Springer Nature 2021
161