Collagen coated μ slides 18 well

For each, 1 μl of a fresh stock of FG domain or variant (1 mM unlabelled protein in 4 M GuHCl, unless specified elsewhere) was rapidly diluted with 50 μl assay buffer, and 7.5 μl of the suspension was mixed with 22.5 μl substrate. As described^{46 (link)}, the substrate contains either 12 μM Hoechst 33342 (Thermo Scientific)/6 μM mCherry/3 μM EGFP/3 μM efGFP_8Q/3 μM efGFP_8R/1 μM of an NTR or [1.5 μΜ NTR pre-incubated with 1 μΜ cargo], in assay buffer. Note: before mixing NTF2 with RanGDP-Atto488, the Ran protein was incubated with 2 μM RanGAP for 30 mins, which converts all Ran to the GDP-bound form in the assay. The resulting mixture was placed on collagen-coated μ-slides 18-well (IBIDI, Germany). FG particles were allowed to sediment under gravity for 1 h before imaging by CLSM. The samples for ODT and CLSM were prepared independently.

A previously described procedure^{32 (link)} was applied with minor modifications. Typically, 2 μl of a 1 mM (≈60 μg/μl) FG domain solution (in 4 M GuHCl) was rapidly diluted with 100 μl assay buffer (50 mM Tris/HCl pH 7.5, 150 mM NaCl, 5 mM DTT) and 7.5 μl of the suspension was mixed with 22.5 μl substrate (typically 1 μM NTRs or 6 μM mCherry in assay buffer, see also below). The resulting mixture ([FG domain] = 5 μM) was placed on collagen-coated μ-slides 18-well (IBIDI, Germany) that had been further passivated with 0.1 mg/mL MBP. Before imaging, FG particles were allowed to sediment for 60 min to the bottom of the slide.