Proteins were purified and assembled into scaffolds in vitro as previously described (Woodruff et al., 2015 (link)). For a detailed protocol please refer to Woodruff and Hyman (2015) (link). In brief, all reactions were carried out in network buffer (25 mM HEPES pH 7.4, 135 mM KCl, 125 mM NaCl, 0.2 mM ATP, 10 mM MgCl2, 1 mM DTT, 0.02% CHAPS, 0.2% glycerol, 0.025 mg/ml ovalbumin) plus pre-blocked 0.2 µm red fluorescent polystyrene beads (Invitrogen) to aid in finding the focal plane. All proteins and reagents were stored on ice prior to being mixed, aliquoted into multiple tubes and then incubated at 23°C. For analysis of a single reaction, 2 µl of that reaction was spotted onto a non-frosted cover slide and then covered with 18×18 mm pre-cleaned hydrophobic cover slips. Statistical analyses were carried out using two-tailed Student's t-test.
Cover slips were cleaned and made hydrophobic using the following steps. First, cover slips were placed in a Teflon holder and submerged in a 1:20 dilution of Mucasol detergent (Sigma) for 10 min with sonication. Second, the cover slips were transferred to 100% ethanol and incubated for 10 min with sonication. Third, the cover slips were incubated in a 50% solution of Rain-X (diluted in ethanol) for >30 min, then washed in ethanol, then twice in water. Finally, the cover slips were dried using N2 gas and stored in a desiccation chamber.
Cover slips were cleaned and made hydrophobic using the following steps. First, cover slips were placed in a Teflon holder and submerged in a 1:20 dilution of Mucasol detergent (Sigma) for 10 min with sonication. Second, the cover slips were transferred to 100% ethanol and incubated for 10 min with sonication. Third, the cover slips were incubated in a 50% solution of Rain-X (diluted in ethanol) for >30 min, then washed in ethanol, then twice in water. Finally, the cover slips were dried using N2 gas and stored in a desiccation chamber.