Friction limits the operation of macroscopic machines. Using optical tweezers, we showed that friction also limits the operation of molecular machines by measuring the friction between single yeast kinesin-8, Kip3p, and its microtubule track. The protein friction arises from the force necessary to break the adhesive bonds that Kip3p forms with discretely, 8-nm spaced binding sites on its track. A model based on bond rupture dynamics with a single energy barrier described the data. A uctuation analysis confirmed Kip3p stepping during diffusion. Here, we validate our experimental results and data analysis by a Monte Carlo simulation. Our data have implications for other molecular machines or actively driven proteins, and give further insight into diffusion of proteins along polymers such as microtubules or DNA.© (2010) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.