2.5. Purified Actin Polymerization Measurement Assay

Polymerization of purified actin was measured by changes in pyrene fluorescence using a fluorescence spectrophotometer (Safas Xenius, Safas SA, Monaco) as described previously [32]. Briefly, actin was purified from rabbit skeletal muscle acetone powder and monomeric Ca²⁺-ATP actin was purified by Sephacryl S-300 chromatography in G buffer (5 mM Tris-HCl, pH 8, 0.2 mM ATP, 0.1 mM CaCl₂, 0.5 mM DTT, 0.1 mM azide). Actin was labeled on Cys-374 with pyrene iodoacetamide. Monomeric Mg²⁺-ATP-10%pyrene-actin is prepared by incubation of Ca²⁺-ATP-actin (9 µM black actin + 1 µM pyrene labeled actin) on ice with a 0.2 mM EGTA and an 11-fold MgCl₂ concentration relatively to actin. Actin (2 µM) is polymerized by the addition of 1/10 KMEI 10× (500 mM KCl, 10 mM MgCl₂, 10 mM EGTA and 100 mM HCl-imidazole, pH 7).