Actin purification

Lyophilized rabbit skeleton muscle acetone powder was rehydrated and ground in G-buffer (5 mM Tris-HCl, pH 8, 0.2 mM ATP, 0.1 mM CaCl₂, 0.5 mM DTT, annd 0.1 mM sodium azide), and then cleared by centrifugation at 27,000 × g in the JA25.50 rotor (Beckman Coulter, Inc., USA). Solubilized actin in the supernatant was collected and polymerized by adding 50 mM KCl and 2 mM MgCl₂ for 1 h, followed by the addition of 0.8 M KCl for 30 min at 4 °C. F-actin was pelleted by centrifugation at 147,600 × g using a Type50.2Ti rotor (Beckman Coulter, Inc., USA) and then depolymerized by brief sonication and dialysis against G-buffer for 48 h at 4 °C. Monomeric Ca²⁺-ATP-actin was cleaned by centrifugation at 193,900 × g for 2.5 h using the SW 55Ti rotor (Beckman Coulter, Inc., USA). The supernatant was collected and loaded to Sephacryl S-300 HR for gel filtration chromatography using G-buffer (5 mM Tris-HCl, pH 8, 0.2 mM ATP, 0.1 mM CaCl₂, 0.5 mM DTT, and 0.1 mM sodium azide) to obtain monomeric Ca²⁺-ATP-actin.