Hydrophobic interaction chromatography (HIC)

A Dionex ProPac HIC − 10 column of 100 × 4.6 mm (ThermoFisher scientific, 063655) was used to rank apparent hydrophobicity. All separations were carried out on an Agilent HP1260 HPLC (Agilent) equipped with a fluorescence detector. The column temperature was maintained at 20°C throughout the run and flow rate was set at 0.8 mL/min. The mobile phase for the HIC method consisted of 0.8 M ammonium sulfate, 50 mM phosphate pH 7.4 or 0.8 M ammonium sulfate, 50 mM sodium acetate pH 5.0 (buffer A) and 50 mM phosphate pH 7.4 or 50 mM sodium acetate pH 5.0 (buffer B). Following a 5 min hold at 0% B, bound protein was eluted using a linear gradient from 0 to 100% B over 45 min and the column was washed with 100% B for 2 min and re-equilibrated in 0% B for 10 min. The separation was monitored by intrinsic fluorescence with excitation occurring at 280 nm and emission at 340 nm.