4.15. Pyrene-Actin Polymerization Assay

Actin polymerization was monitored by the increase in fluorescence of 20% pyrenyl-actin at 407 nm (excitation at 365 nm) in a fluorescence spectrometer (Jasco FP-8500, Gross-Umstadt, Germany). The final concentration of actin in the reaction was 4 µM. DRR1 proteins were added to G-actin in a constant volume and polymerization was induced by the addition of 1:10 volume of 10× F-buffer (250 mM Tris-HCl pH 7.4, 250 mM KCl, 40 mM MgCl₂, 10 mM EGTA, 10 mM ATP, 10 mM DTT). The polymerization was monitored for 1 h at 21 °C with a cycle interval of 5.5 s.