Fig. 2: Phase separation of three different proteins induced by the pH jump approach.

LLPS of TDP-43 LCD (a, d, and g), NUP98 LCD (b, e, and h) and full-length ERD14 (c, f, and i) was induced by a pH jump from pH = 3.5 to 7.5 (TDP-43 LCD, at 80 μM), pH = 3.0 to 7.5 (NUP98 LCD, at 10 μM) and pH = 11.0 to 6.6 (ERD14, at 20 μM in the presence of 1 mg/ml poly(U) and 8% PEG 6000). Droplets were visualized by fluorescence microscopy of Dylight 488-labeled proteins (mixed into ×200 excess of non-labeled proteins, a, b, and c) without and with salt (150 mM NaCl) immediately after initiating LLPS (marked 0 h) and after 2 h of incubation. LLPS was also monitored by turbidity (OD600) measurement (d, e, and f) in the absence of NaCl (blue lines) and in the presence of 150 mM NaCl (green lines). The size evolution of droplets formed upon LLPS was also followed by DLS (g, h, and i), in the absence of NaCl (blue lines), and in the presence of 150 mM NaCl (green lines, fitting functions are also shown on panels). All kinetic traces are mean ± SD of experiments in triplicate (n = 3).