Kif7 Binding to Taxol-Stabilized Microtubules

GDP-taxol stabilized microtubules were prepared from purified, pre-cleared bovine tubulin as described in Subramanian et al., 2010^{90 (link)}. Kif7-DM-GFP (1 μM) was incubated with microtubules (0–10 μM) in the presence of 1mM ATP, 2mM MgCl₂, with or without 5μM Gli for 15 minutes at room temperature in 1x BRB80 supplemented with 0.1 mg/mL BSA and then subjected to sedimentation by ultracentrifugation at 90,000 x g for 10 minutes at 27°C in a TLA 120.1 rotor (Beckman Coulter). Pellets were subsequently resuspended and the amount of protein in each pellet and supernatant was analyzed by SDS-PAGE. Proteins were stained using Gel Code^™ Blue Stain (Thermo Fischer Scientific) and scanned using GE Typhoon FLA 9000 Gel Scanner. Dissociation constants were calculated by fitting of the experimental data to a Hill equation using GraphPad Prism.