Extended Data Fig. 2: Controls for in vitro phase separation assays.
From: Assembly of synaptic active zones requires phase separation of scaffold molecules
a, In vitro LLPS assay workflow and scoring conditions. b, Size-exclusion chromatography of purified mCherry and mCherry–FUS in 20 mM Tris pH 7.4, 500 mM NaCl, indicating that the protein isolated by the high-salt purification method is soluble. A280, absorption at 280 nm. c, In vitro LLPS assay. Left panels, soluble input materials before dilution to physiological salt concentration. Right panels, comparison between the effects of no crowding and 10% PEG conditions on a known phase-separating motif (FUS)41 and a negative control (mCherry). d, Representative FRAP images (top) and quantified FRAP dynamics (bottom) of FUS and mCherry in vitro condensates formed in 10% PEG. Data are means ± 95% CI from three independent experiments. e, Time-lapse analysis of in vitro condensates containing mCherry and FUS, showing the presence or absence of liquid behaviour. Phase-separation assays were performed in 20 mM Tris pH 7.4, 150 mM NaCl and 10% PEG 3350 where indicated with 10 μM protein. f, g, Phase diagrams for mCh–SYD-2(Nter) (f) and mCh–ELKS-1 (g), showing protein concentrations versus NaCl or PEG concentrations. Each combination was tested three times. Scale bars, 5 μm.
