D2-mCherry in imaging buffer (BRB80, 25% glycerol 1× blocking solution, 0.1% methylcellulose, 5 mM protocatechuic acid (Pacific Bioscience), 5 mM TSY (Pacific Bioscience), and 50 nM protocatechuate-3,4-dioxygenase (Pacific Bioscience)) was applied to the microtubule-immobilized glass chamber (see the Preparation for binding assay section). After 5 min of incubation, image acquisition was conducted for all registered positions. To increase the signal-to-noise ratio, five frames were taken for every position and channel.
For assays in the presence of taxol, D2-mCherry in imaging buffer was supplemented with 20 μM taxol, and images were acquired the same way. Taxol depletion was conducted by exchanging the solution with a sample of same D2 concentration without taxol. Images were taken after 5-min incubation.
Because the binding activity of D2 was lost quickly after being thawed (<1 h), we prepared new samples every 30 min.
For assays in the presence of taxol, D2-mCherry in imaging buffer was supplemented with 20 μM taxol, and images were acquired the same way. Taxol depletion was conducted by exchanging the solution with a sample of same D2 concentration without taxol. Images were taken after 5-min incubation.
Because the binding activity of D2 was lost quickly after being thawed (<1 h), we prepared new samples every 30 min.
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