(A) Schematic of single-molecule motility assays on surface-immobilized MTs decorated with streptavidin-coated QD585 obstacles. Human kinesin-1, yeast dynein, and mammalian DDB are labeled with QD655 at their tail domain. (B) Example fluorescent images of QD585 obstacles on MTs at different QD concentrations. (C) The linear density of QDs on MTs at different QD concentrations (mean ± SD, from left to right n = 97, 98, 104 and 90 MTs from two technical replicates). (D) Kymographs show the motility of QD655-labeled motors on MTs with or without QD obstacles. The QD585 signal is not shown. (E) Mobile fraction, velocity and run length for all three motors were all normalized to the no QD condition (mean ± SD, three independent experiments). Run-length values represent decay constants derived from a single exponential decay fit. From left to right, n = 271, 423, 405 for kinesin, 315, 407, 197, 168 for yeast dynein, and 636, 502, 356, 509 for DDB.