Covalent Immobilization of Microtubules on Glass Surfaces for Molecular Motor Force Measurements and Other Single-Molecule Assays

Rigid attachment of microtubules (MTs) to glass cover slip surfaces is a prerequisite for a variety of microscopy experiments in which MTs are used as substrates for MT-associated proteins, such as the molecular motors kinesin and cytoplasmic dynein. We present an MT-surface coupling protocol in which aminosilanized glass is formylated using the cross-linker glutaraldehyde, fl uorescence-labeled MTs are covalently attached, and the surface is passivated with highly pure beta-casein. The technique presented here yields rigid MT immobilization while simultaneously blocking the remaining glass surface against nonspecifi c binding by polystyrene optical trapping microspheres. This surface chemistry is straightfor-ward and relatively cheap and uses a minimum of specialized equipment or hazardous reagents. These methods provide a foundation for a variety of optical tweezers experiments with MT-associated molecular motors and may also be useful in other assays requiring surface-immobilized proteins