Cfx96 touch thermal cycler cfx

Purified CAPN5-PC samples (1 mg/mL) were analyzed by differential scanning fluorimetry (DSF) to test the ability of added calcium to bind and alter its thermal stability. Assays were performed on a Bio-Rad CFX96 Touch Thermal Cycler-CFX real-time-PCR system using the FRET channel and contained 300 mM NaCl, 20 mM HEPES (pH 7.5), 2 mM DTT, 5% glycerol, and 5x SYPRO-Orange dye (Invitrogen). Temperature was increased from 1°C /min from 4°C to 100°C in the presence of increasing concentrations of added CaCl₂ (0.5 to 100 mM). As the temperature rose, the protein unfolded to expose hydrophobic residues to the SYPRO-Orange dye, leading to an increase in fluorescence signal. The temperature at which the protein starts to unfold is called the melting temperature (T_melt). Fluorescence traces were analyzed with DMAN software (Wang et al., 2012 (link)).