Figure 3

Optimization of room temperature stability of CHP3 for MST analysis. (a) Thermal stability of CHP3 in dependence of pH was analyzed by nanoDSF. CHP3 samples were diluted in succinic acid/sodium phosphate monobasic monohydrate/glycine buffers of defined pH (pH values are listed in the color code inset). A control buffer was used at physiological pH (20 mM Hepes pH = 7.2, 150 mM NaCl). Changes in fluorescence upon increasing temperature were monitored and the first derivative of the fluorescence ratio at 350 nm and 330 nm (F350/F330) is shown (one representative curve from three measurements). At and below pH 5, CHP3 melting curves indicated aggregation and lacked a defined melting point. Above, the melting points range from 61.1 to 64.3 °C. The melting point of 63.6 °C of the control buffer is indicated as dashed line. (b) Thermal stability of CHP3 was analyzed in MST labelling and measuring buffer probing the effect of supplementation with DMSO, Ca²⁺ and Mg²⁺. The following melting points were determined: labelling buffer, 63.9 °C; labelling buffer with 7% DMSO 61.7 °C; measuring buffer with MgCl₂ 67.1 °C; measuring buffer with CaCl₂ 63.4 °C.