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4 G = H-TS and the equations (2.2) and (4), where R is the universal gas constant, T the reaction temperature, ΔG the change in Gibbs free energy, ΔH the change in enthalpy and ΔS the change entropy. Substituting (4) into (2.2) yields Plotting ln (K a ) (in molar concentrations) against 1/T yields the so called van´t Hoff plot. ΔH can be obtained from the slope m of the linear fit (Fig.3 B) as Under the assumption that ΔH is constant in the relatively small linear range of the van´t Hoff plot (Figure 3) it is also possible to directly derive ΔS from the plot as The universal gas constant R was converted from SI units to [cal/mol *K]. An example of how to derive a linear fit of the van´t Hoff plot from MST measurements is shown in Figure 3. Figure 3: A) Van´t Hoff plot of a complete set of K a values of the template-PM interaction for illustration of the data point distribution. Note that the divergence of the ln (K a ) values becomes high at low temperatures due to very high affinities (K a >10 9 ) and thus a larger margin of error in K d measurements. B) Linear range of the van´t Hoff plot and corresponding linear fit. General comments on quality control Once the entire temperature gradient was measured by MST, one additional experiment using the same capillaries at the starting temperature (24 °C) was performed as a quality control: Both fluorescence intensities and MST signal of this experiment was identical to the first experiment of the temperature gradient series, ensuring the validity of this approach. If this is not the case, the labeled biomolecules might degrade over time. In this case, measurement in overlapping intervals is recommended. For this, fresh solutions are prepared and measured in small intervals of 6 °C whereby 2 increments are overlapping. If the resulting timetraces still differ, the molecules might not tolerate the temperature gradient and/or acquisition times, which require optimization of buffer and experimental conditions to stabilize the molecules. References http://biophysics.idtdna.com/ © 2013 NanoTemper Technologies GmbH K d = 1 K a ( 3) ln ( K a )= −ΔG RT (2.2) ln ( K a )= −ΔH RT + ΔS R (5) m= −ΔH R y (0)= ΔS R