Kinesin, Microtubule, and Actin Assay

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 previously^{9 (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.