We tested several parameters that might affect the formation of liquid-like droplets during LLPS process, including protein concentration, storage temperature, ionic strength, and pH value. All the experiments were carried out immediately after protein purification, and the formation of liquid-like droplets in protein solutions was monitored on the basis of turbidity measurement. To test the influence of protein concentration on the formation of liquid-like droplets, we applied fresh purified TLC-M solutions with varied concentrations (0.2, 0.5, 1, and 2 mg/ml) incubated at 4 and 25°C to measure the turbidity over time.

Further, to test the influence of temperature on the formation of liquid-like droplets, we measured and compared the turbidity of TLC-M solutions (1 mg/ml) after dialyzing in dialysate buffers [20 mM tris-HCl, 50 mM NaCl, and 0.5 mM EDTA (pH 5)] at 4, 16, and 25°C for 1 hour. To assess the influence of ionic strength (varied NaCl concentration) on the formation of liquid-like droplets, we measured and compared the turbidity of TLC-M solutions (1 mg/ml) after dialyzing in dialysate buffers [20 mM tris-HCl and 0.5 mM EDTA (pH 5)] containing different NaCl concentrations (0.05, 0.2, 0.5, and 1.0 M) for 1 hour. To assess the influence of solution pH, we measured and compared the turbidity of TLC-M solutions (1 mg/ml) after dialyzing in dialysate buffers (20 mM tris-HCl, 50 mM NaCl, and 0.5 mM EDTA) at different pH values (3, 5, 7, 9, and 11) for 1 hour. All data are presented in fig. S5.

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