Thermodynamics Measurements
Overview
Thermodynamic measurements are crucial for determining enthalpic (ΔH) and entropic (ΔS) contributions in molecular interactions. By measuring the dissociation constant (Kd) at different temperatures, we can directly observe how binding affinity varies with temperature. Van't Hoff analysis of these Kd values then allows determination of ΔH and ΔS, providing deeper thermodynamic insights.
The thermodynamic measurement is performed with spectral shift data and therefore this mode is available exclusively in MO.Control 2 for Monolith X. It is not supported in Expert Mode. While this guide discusses setup and software handling, the Van't Hoff Analysis article goes into more detail on theoretic background and data analysis.
Experimental Setup
The thermodynamic experiment is conducted over an adjustable temperature range between 20°C and 35°C. Temperature increments must be set between 1°C and 4°C, with a minimum of four temperature points to ensure valid analysis. These parameters are critical for accurately calculating thermodynamic constants. Insufficient temperature data points will prevent the experiment from proceeding.
Each temperature point requires an incubation step for 1 to 90 minutes (see Figure 1), based on user-defined requirements and dependent on how quickly an interaction equilibrates at a new temperature. As a rough guidance, small molecule – protein interactions typically equilibrate very fast, while protein – protein interactions, in particular high-affinity antibody – antigen interactions, can require more time to adjust to a new equilibrium. Fluorescence shifts are detected using the Ratio 670nm/650nm mode (Spectral Shift), optimized for far-red fluorophores, which enhances sensitivity for detecting ligand-binding events.
Figure 1. Example settings for Thermodynamics Measurements. The Thermodynamic Setup is located in the "Plan" tab of MO.Control 2.
Both standard and premium capillaries can be used for thermodynamic experiments. The setup supports series types with either 12 or 16 capillaries, providing flexibility depending on experimental needs.
Data Acquisition and Export
Binding affinities are measured at different temperatures, with the natural logarithm of the equilibrium constant (ln(Kd)) plotted against the reciprocal temperature (1/T, in Kelvin). If enthalpy and entropy are assumed to be temperature-independent, this results in a linear Van't Hoff plot. Any deviation from linearity may indicate complex thermodynamic behaviors, such as changes in heat capacity (ΔCp) or shifts in reaction mechanisms. The following parameters will be acquired:
- Enthalpy (ΔH): Derived from the slope of the Van't Hoff plot.
- Entropy (ΔS): Estimated from the y-intercept of the plot.
Specific temperature points or capillaries can manually be in- or excluded by clicking the respective boxes to and refine the analysis if irregularities are found (see Figure 2).
Raw data can be exported in CSV format and Van't Hoff plots as PNG images for further analysis. The export button is located above the plots on the data page.
Figure 2. "Data" tab of a Thermodynamics Measurement in MO.Control 2 where the fitted parameters are displayed, and outlier selection and data export are possible.