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To quantify the amount of surface-bound GFP-kinesin via fluorescence measurements, we used silicon wafers with a 75-nm oxide layer (obtained from GeSiM mbH, Rossendorf) in order to avoid destructive interference effects present at low oxide thickness.
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Before usage, the coverslips were immersed in a PEG solution that consisted of 5 mM 2-[methoxypoly-(ethyleneoxy)propyl]-trimethoxysilane (90%, ABCR Karlsruhe) and 0.08% hydrochloric acid (36%) in toluene. After 12 h, the coverslips were rinsed and sonicated in toluene, ethanol, and water. The PEG layer on the glass coverslip prevented kinesin and microtubule binding to that side of the flow cell.
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2, 1 mM Mg-GTP, and 5% DMSO at 37 °C. After 30 min, the microtubule polymers were stabilized and 100-fold diluted in room-temperature BRB80 containing 10 μM taxol. Microtubules were sheared by passing them up and down three times through a 30-gauge needle.
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Although, using the presented method the fraction of the short microtubules would always contain a number of long ones, this could potentially be overcome by flowing the microtubules into the device at high concentration. Because of a higher diffusion coefficient, shorter microtubules would reach the active kinsein binding sites faster than the longer ones. Having bound to the surface at high density, the short microtubules would then prevent the binding of the longer ones because of steric hindrance (note that microtubules are stiff polymers with a persistence length of about 1 mm in solution and that they have to land rather parallel to the surface in order to attach to the kinsein motors).
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