Peaq dsc analysis software

DSC experiments were performed with the PEAQ Differential Scanning Calorimeter Automated (Malvern Panalytical); 80 µM of the respective binder in PBS were heated up from 20 °C to 110 °C with a heating rate of 1 °C/min. Data analysis was performed with the PEAQ-DSC analysis software (Malvern Panalytical). After buffer baseline subtraction and normalization for protein concentration, transitions were fitted with a non-two-state thermal unfolding model.

To achieve identical buffer composition for reference and sample cells every sample was buffer exchanged using a Superdex‐200 5/150 GL or Superdex‐200 10/300 Increase column (GE Healthcare, Chicago, Illinois) connected to an HPLC 1260 Infinity with fraction collector (Agilent Technologies, Santa Clara, CA).
DSC experiments were performed with the PEAQ Differential Scanning Calorimeter Automated (Malvern Panalytical, Malvern, UK). Experiments were performed under increased pressure (~62 psi) to prevent the solutions from boiling. At the beginning of an experiment, at least four buffer runs (i.e., buffer in both sample and reference cells) were performed to establish the thermal history of the cells and to collect optimal buffer scans for buffer subtraction.
A temperature slope of 1°C/min was used for all experiments. Upon scanning from 20°C to 130°C and reheating again, we did not encounter any protein refolding. Therefore, the reheated run (i.e., rescan) was taken for buffer subtraction. Data analysis was performed with the PEAQ‐DSC analysis software (Malvern Panalytical, Malvern, UK). The baseline was fitted with the spline baseline correction model to account for differences in the heat capacities of the folded and unfolded states of the protein. Concentration normalization was performed and transitions were fitted with a two‐state thermal unfolding model.