Single-molecule microtubule dynamics assay

Chamber preparation and microscopy were performed as described (Volkov et al., 2018 (link)). In brief, both coverslips and slides were cleaned in oxygen plasma, immediately silanized, and later assembled in a chamber using double-sided tape. The chambers were first incubated with ~0.2 µM anti-DIG antibody (Roche) and passivated with 1% Pluronic F-127, followed by GMPCPP seeds (diluted 1:200 – 1:1000) and then the reaction mix. The reaction mix contained MRB80 buffer supplemented with 8 µM tubulin (4–6% labeled with HiLyte-488), 1 mM GTP, 1 mg/ml κ-casein, 0.01% methylcellulose, 4 mM DTT, 0.2 mg/ml catalase, 0.4 mg/ml glucose oxidase and 20 mM glucose; this mix was centrifuged in Beckman Airfuge for 5 min at 30 psi before adding to the chamber.
Imaging was performed at 30°C using Nikon Ti-E microscope (Nikon) with the perfect focus system (Nikon) equipped with a Plan Apo 100 × 1.45 NA TIRF oil-immersion objective (Nikon), iLas² ring TIRF module (Roper Scientific) and a Evolve 512 EMCCD camera (Roper Scientific). Images were acquired with MetaMorph 7.8 software (Molecular Devices). The final resolution was 0.16 µm/pixel. The objective was heated to 34°C by a custom-made collar coupled with a thermostat, resulting in the flow chamber being heated to 30°C. All images were analyzed using Fiji (Schindelin et al., 2012 (link)).