Biotin–kinesin and MTs stabilized with GMP-CPP (Guanosine-5'-[(α,β)-methyleno]triphosphate) were obtained from Brandeis University’s National Science Foundation Materials Research Science and Engineering Center and stored at –80 °C. Kinesin, once thawed, was kept on ice, while MTs were kept at room temperature. Actin was purified from rabbit skeletal muscle as described previously9 (link). No rabbits were directly involved in the study. Monomeric actin was stored at 4 °C in the so-called G-Buffer (2 mM Tris, 0.2 mM ATP, 0.2 mM CaCl2, 0.2 mM DTT and 0.005% NaN3 at pH 8.0). Gelsolin was purified from adult bovine serum (Sigma-Aldrich). Alexa Fluor 488 phalloidin, streptavidin and Texas Red DHPE dye were purchased from Thermo Fisher. The remaining lipids (l-α-phosphatidylcholine (egg PC) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(biotinyl(polyethylene glycol)-2000]) (DSPE–PEG(2000)–biotin, where 2,000 is the PEG molecular weight)) were purchased from Avanti and stored in chloroform. Methylcellulose, creatine phosphate, creatine phosphokinase, catalase and pyranose oxidase were purchased from Sigma.
Free full text: Click here