Technical Notes

Sealing-free measurements

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Technology Brief 4 nanoDSF Sealing-free measurements With the Prometheus instrument One major benefit of the Prometheus instruments is that they acquire high-quality thermal stability data of proteins using a minimal number of handling steps. Specifically, the patented capillary format (DE102014018535A1) allows for sample loading without pipetting steps, simply by dipping the capillaries into the protein solution. Moreover, the design of the thermal element in combination with the capillary sample format enables measurements of thermal unfolding curves with unsealed capillaries, which means that no additional handling steps are required between the filling of the capillary with the sample and loading it into the instrument. This makes the Prometheus instrument one of the most simple, straightforward and time- saving analytical instruments available. To demonstrate that sealing of capillaries is not required for thermal unfolding experiments, 6 different proteins / compounds were tested side-by-side in sealed and non-sealed capillaries. Reference capillaries were sealed using the Capillary Sealing Kit (PR-P001). For this comparative study, 2 mAbs, 3 globular proteins with different sizes, and the amino acid tryptophan were used to cover a large range of molecular weights, molecular sizes and thus diffusion coefficients. The thermal unfolding experiment was performed using a heating rate of 1 °C/min from 20 °C to 95 °C. The fluorescence ratio values F350/F330 show that the unfolding curves of the proteins in sealed (red) and non-sealed (green) capillaries overlay perfectly, as shown in Figure 1. Moreover, the respective fluorescence intensities over time are also the same, demonstrating that no sample concentration occurs during the course of the experiment. The identical signals in sealed and non-sealed capillaries also show that the Capillary Sealing Paste does not influence the unfolding signal. A second experiment using a long-term isothermal measurement was performed to demonstrate when capillary sealing is recommended. For this, 48 identical samples were measured, 24 in sealed and 24 in non-sealed capillaries. The measurement was conducted at 40 °C for a time period of 15 hours. Figure 2 shows that the F330 fluorescence stays equal in the 24 sealed capillaries over the entire measurement time of 15 hours. In some of the non-sealed capillaries however, an increase in the F330 fluorescence can be observed after ~ 300 minutes, which relates to a concentration increase due to evaporation. Prior to 300 minutes however, the fluorescence signals from the sealed and non-sealed capillaries are identical. We therefore recommend to use non-sealed capillaries for measurements < 180 minutes. Capillary sealing should be used for experiments that require longer measurement times.

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