Motor-driven cytoskeletal filaments are versatile transport platforms for nanosized cargo in molecular sorting and nano-assembly devices. However, because cargo and motors share the filament lattice as a common substrate for their activity, it is important to understand the influence of cargo-loading on transport properties. By performing single-molecule stepping assays on biotinylated microtubules we found that individual kinesin-1 motors frequently stopped upon encounters with attached streptavidin molecules. Consequently, we attribute the deceleration of cargo-laden microtubules in gliding assays to an obstruction of kinesin-1 paths on the microtubule lattice rather than to 'frictional' cargo-surface interactions. We propose to apply this obstacle-caused slow-down of gliding microtubules in a novel molecular detection scheme: Using a mixture of two distinct microtubule populations that each bind a different kind of protein, the presence of these proteins can be detected via speed changes in the respective microtubule populations.