The Optical Trapping Dumbbell Assay for Nonprocessive Motors or Motors That Turn around Filaments

Abstract

In vitro motility assays enabled the analysis of coupling between ATP hydrolysis and movement of myosin along actin filaments or kinesin along microtubules. Single-molecule assays using laser trapping have been used to obtain more detailed information about kinesins, myosins, and processive DNA enzymes. The combination of in vitro motility assays with laser-trap measurements has revealed detailed dynamic structural changes associated with the ATPase cycle. This protocol describes the preparation of biotin–actin filaments and coverslips coated with polystyrene beads. These are then used in optical trapping dumbbell assays to study interactions between motors and filaments.