Pyrene actin polymerization assays

G-actin (1 mg/ml; tebu-bio, Cytoskeleton) and pyrene-labelled G-actin (0.1 mg/ml; tebu-bio, Cytoskeleton) were incubated on ice for 1 hour and centrifuged at 4 °C for 20 min at 14000 g. To analyse actin polymerization, 40 µl Drebrin wt or DrebrinS142D (50 ng/µl), 5 µl EB3 (100 ng/µl), 5 µl ATP (5 mM), 5 µl GTP (20 mM), 10 µl taxol (200 µM), 10 µl MTs prepared as indicated earlier, 5 µl G-actin (1 mg/µl) and 10 µl pyrene labelled G-actin (0.1 mg/ml) were added to microtube, mixed and transferred to a 96-well plate. Pyrene fluorescence was monitored via TecanSaphire 2 reader at excitation level of 365 nm with Tecani-control 1.5 and after ten cycles 10x polymerization buffer (50 mM Tris-HCl, pH 7.5, 500 mM KCl, 20 mM MgCl₂, 10 mM EGTA, 2 mM ATP) was added to start polymerization. Subsequently pyrene fluorescence was monitored for further 30 min.