Measuring Chloride Binding Affinity of CLC-ec1 F357A

Cl^- binding affinity was determined for purified CLC-ec1 F357A as described (Picollo et al., 2009 (link); Basilio et al., 2014 (link)) using a nanoITC instrument (TA Instruments). For these experiments, the final purification step of the protein was purified over a gel filtration column pre-equilibrated in 100 mM Na-K-Tartrate, 20 mM Hepes, 50 μM DMNG, pH 7.5 (Buffer B0) and concentrated to 50–195 μM. The injection syringe was filled with buffer B0 with 50 mM KCl added, to achieve final Molar Ratios of 70–100. Each experiment consisted of 30–48 injections of 1 μl of the ligand solution at 3–4 min intervals into the experimental chamber kept under constant stirring at 350 rpm and at 25.0 ± 0.1°C. All solutions were filtered and degassed prior to use. The ITC data was fit to a single site Wiseman isotherm using the NanoAnalyze program from TA instruments.