Motility assays are the tools of choice for the studies regarding the motility of protein molecular motors in vitro. Despite their wide usage, some simple, but fundamental issues still need to be specifically addressed in order to achieve the best and the most meaningful motility analyses. An analysis of the errors in the calculation of the average velocity and of the fluctuations of velocity due to pixel size is presented here. The magnitude of the fluctuations is correlated with the resolution of the objective lens used in observing the motility assays and with the parameters of the camera used for digitizing the images. Also, the errors in the angular distribution of velocities due to pixel size are characterized and discussed.
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