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 ofmicroscopy experiments in which MTs are used as substrates for MT-associated proteins, such as themolecular motors kinesin and cytoplasmic dynein. We present an MT-surface coupling protocol in whichaminosilanized glass is formylated using the cross-linker glutaraldehyde, fl uorescence-labeled MTs arecovalently attached, and the surface is passivated with highly pure beta-casein. The technique presentedhere yields rigid MT immobilization while simultaneously blocking the remaining glass surface againstnonspecifi c binding by polystyrene optical trapping microspheres. This surface chemistry is straightforwardand relatively cheap and uses a minimum of specialized equipment or hazardous reagents. Thesemethods provide a foundation for a variety of optical tweezers experiments with MT-associated molecularmotors and may also be useful in other assays requiring surface-immobilized proteins.