Purification of mEGFP and EB1-GFP

To obtain mEGFP, 0.5 mg of GFP-γ-TuNA (Wieczorek et al., 2020b (link)) was incubated with 0.5 mg of PreScission protease (Cytiva) on ice for 2 h. The reaction was gel-filtered over a Superdex 75 10/300 column (Cytiva) preequilibrated in 40 mM Hepes, pH 7.5, 150 mM NaCl, 1 mM MgCl₂, and 2 mM 2-mercaptoethanol. Purified mEGFP was supplemented with glycerol to 10% (vol/vol), snap-frozen in liquid N₂, and stored at −80°C. EB1-GFP in a pET-DUET vector (Novagen) was expressed and purified from BL21(DE3) Rosetta (Novagen) cells using Ni-NTA affinity followed by gel filtration using the methods described in Forth et al. (2014) (link).