Protein Purification and Droplet Assay

For protein purification, Plasmids containing the protein of interest fused to GFP were transformed into BL21 cell (NEB, C2527I). A fresh bacterial colony was inoculated into 200 ml LB media containing kanamycin and grown at 37 °C until an OD₆₀₀ of 0.8–0.9 has been reached. IPTG was added to 1 mM and growth continued overnight at 16°C. Pallets from 200 ml cells were resuspended in 10 ml of GST lysis buffer (50 mM Tris-HCl pH7.5, 100 mM NaCl) containing 1 mM dithiothreitol, 0.2 mM Phenylmethylsulfonyl fluoride, 1% Triton-X100, complete protease inhibitor and sonicated (4 cycle of 30 sec on, 30 sec off). The lysate was cleared by centrifugation at 12,000 × g for 20 min at 4 °C and added NaCl to 500 mM. Then the supernatant was added 500 μl glutathione agarose (Thermo Fisher, 16100) (prewashed in lysis buffer). Tubes containing this agarose lysate slurry were rotated at 4 °C overnight. Then the packed agarose washed twice with GST lysis buffer containing 500 mM NaCl and twice with GST lysis buffer. Protein was obtained by cleavage of Pierce^TM HRV 3 C protease and judged by coomassie stained gel.
For droplet assay, protein was added to solutions at varying concentrations with indicated final salt and molecular crowder concentrations in Buffer A (50 mM Tris-HCl pH 7.5, 10% glycerol, 1 mM DTT). The protein solution was immediately loaded onto a homemade chamber comprising a glass slide with a coverslip attached by two parallel strips of double-sided tape. Slides were then imaged with an Andor confocal microscope with a ×100 objective. Imaging was obtained by the Olympus FV1000 IX81-SIM Confocal Microscope (Olympus, Tokyo, Japan).