Superose 6 5 150 gl

Approximately 9 μg of purified human PKD2 channel proteins (WT or C331S) in 30 μL buffer composed of 20 mM Hepes, 150 mM NaCl, 2 mM CaCl₂, 0.5 mM TCEP, and 0.5 mM DDM, at pH 7.4, were incubated at 4 °C to 90 °C (4 °C, room temperature, 30 °C, 35.2 °C, 39.3 °C, 44.9 °C, 49 °C, 54 °C, 60 °C, 65.5 °C, 69.4 °C, 75 °C, 79.3 °C, 84 °C, and 90 °C) for 10 min in a thermal cycler. To reduce the C331–C344 disulfide bond in the PKD2 channel, 10 mM GSH was added to all of the buffers during purification and thermal stability assay. The treated channel samples were then diluted 10 times with the same buffer followed by centrifugation for 30 min at 40,000 rpm. Thirty microliters of each cleared channel samples were separated on an analytical size-exclusion column (Superose 6 5/150 GL; GE Healthcare) at 0.3 mL/min flow rate. Proteins were detected by tryptophan fluorescence. The fluorescence-detection size-exclusion chromatography-based thermostability experiments were performed in triplicates for each temperature point. The integrated area of the channel tetramer peak at different temperature points is normalized to that at 4 °C to generate the thermal stability plot.