Microtubule Immobilization for Microscopy

The assay was performed following a previously described protocol with some changes (Roostalu et al., 2015 (link)). Flow chambers were incubated with 5% Pluronic F-127 in water (Invitrogen, P6866) for 10 min at room temperature and then washed with assay buffer (80 mM Pipes, 30 mM KCl, 1 mM EGTA, 1 mM MgCl₂, 1 mM GTP, 5 mM 2-mercaptoethanol, 0.075% (w/v) methylcellulose (4000 cP; Sigma-Aldrich, M0512), 1% (w/v) glucose, 0.02% (v/v) Brij-35 (Thermo Scientific, 20150)) supplemented with 50 μg mL⁻¹ k-casein (Sigma-Aldrich, C0406) and extra 0.012% (v/v) Brij-35. Flow chambers were then incubated with assay buffer containing 50 μg mL⁻¹ NeutrAvidin (Invitrogen, A2666) for 3 min on a metal block on ice and subsequently washed with BRB80 (80 mM Pipes, 1 mM EGTA, 1 mM MgCl₂). Next, flow chambers were incubated for 5 min at room temperature with biotin- and Alexa-568-labeled GMPCPP-stabilized microtubules diluted 1:2000 in BRB80. Unbound microtubules were removed by subsequent BRB80 washes.