Molecular motors perform work in cells by moving in an ATP-dependent manner along filamentous tracks. In vitro, the mechanical action of such motor proteins can be investigated by attaching the molecules to surfaces in the so-called gliding or bead assays. Surface attachment protocols have to be used that do not interfere with the function of the molecule. Here, we describe a sandwich protocol that preserves functionality. The protocol can be used for a large variety of proteins, in particular kinesin motor proteins that are GFP-tagged.