A Quantitative Comparison of Blocking Agents in the In Vitro Motility Assay

Blocking agents are used to prevent non-specific binding and stabilize motor proteins, both in prospective nano-scale devices and in vitro assays of motor function. Sacrificial proteins such as bovine serum albumin and casein are typically used. However, these may introduce unwanted nonspecific interactions, and may also not be appropriate for all experimental or device applications. We therefore compared actomyosin function in the motility assay when blocked with proteins and synthetic polymers. Polyvinylpyrrolidone (PVP) preserved higher actin filament velocities and motile fractions than traditional protein blocking agents, while polymers based on polyethylene glycol had a deleterious effect. PVP also supports motility by other motor systems. The data suggest a model in which differential preservation of actomyosin function by blocking agents results from myosin adsorbing to surfaces in different orientations.