Preparation of Protein Samples for PS Assays

For PS assays in vitro, the imaging chambers were prepared following the protocol described previously (64 (link)). Briefly, the coverslip was attached to the glass slide using paralleled double-sided tapes, and then small amounts of the protein samples were put in the gap between coverslip and glass slide. Before droplet formation, the concentrated proteins were experienced buffer exchange by centrifugal filtering and concentration determination using BCA Protein Assay kit. The proteins were diluted to a final concentration of 5 μM in a PS buffer (50 mM Tris–HCl, 120 mM NaCl, 2 mM MgCl₂, 10% PEG 8000, pH 7.5) and incubated for 30 min at room temperature, followed by centrifugation at 12,000 rpm for 3 min to remove any aggregates. For dA50-induced PS, the protein samples (5 μM) were incubated with an indicated concentration of dA50 and/or 10% Cy5-labeled dA50 in the PS buffer and incubated for 30 min at room temperature. Then, the protein samples were put onto imaging chambers and visualized by confocal microscopy (Leica TCS SP8 WLL).