The fluorescence-detection size-exclusion-chromatography-based thermostability (FSEC-TS) assay60 (link) was performed to evaluate ion binding to proteins. In this assay, the types of ions in the sample solution were varied and heated to various temperatures, and the thermal stability was evaluated by the decay in peak intensity of the SEC profile.
Purified samples of HcKCR1 WT and KR2 were treated with ions by dialysis (buffer: 0.03% DDM, 20 mM Tris-HCl (pH 8.0), and either 100 mMNaCl, KCl, or CsCl, respectively). Purified HcKCR1 WT and KR2 protein at 0.1 mg mL−1 were separated into 60 μL aliquots in PCR tubes, heated at a range of temperatures from 20°C to 95°C for 15 min, and centrifuged at 20,380 g for 15 min. Then, 20 μL of the supernatant was loaded onto a TSKgel 3000 SWXL column (TOSOH bioscience) equipped with Prominence (Shimadzu), pre-equilibrated with DDM SEC buffer (0.03% DDM, 100 mM NaCl, 20 mM HEPES pH 7.5), and run at a flow rate of 0.6 mL min−1. The experiment was performed in triplicate. The peak heights of 280 nm absorbance were normalized to that from samples incubated at 4°C, and the data were fit to a 4-parameter logistic curve using the GraphPad Prism software.
Purified samples of HcKCR1 WT and KR2 were treated with ions by dialysis (buffer: 0.03% DDM, 20 mM Tris-HCl (pH 8.0), and either 100 mMNaCl, KCl, or CsCl, respectively). Purified HcKCR1 WT and KR2 protein at 0.1 mg mL−1 were separated into 60 μL aliquots in PCR tubes, heated at a range of temperatures from 20°C to 95°C for 15 min, and centrifuged at 20,380 g for 15 min. Then, 20 μL of the supernatant was loaded onto a TSKgel 3000 SWXL column (TOSOH bioscience) equipped with Prominence (Shimadzu), pre-equilibrated with DDM SEC buffer (0.03% DDM, 100 mM NaCl, 20 mM HEPES pH 7.5), and run at a flow rate of 0.6 mL min−1. The experiment was performed in triplicate. The peak heights of 280 nm absorbance were normalized to that from samples incubated at 4°C, and the data were fit to a 4-parameter logistic curve using the GraphPad Prism software.