title: Covalent immobilization of microtubules on glass surfaces for molecular motor force measurements and other single-molecule assays.
abstract: 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, fluorescence-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 nonspecific binding by polystyrene optical trapping microspheres. This surface chemistry is straightforward 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.
authors.affiliation: Department of Anatomy and Structural Biology, Gruss Lipper Biophotonics Center, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY, 10461, USA.
authors.forename: Matthew P
authors.initials: MP
authors.lastname: Nicholas
authors.forename: Lu
authors.initials: L
authors.lastname: Rao
authors.forename: Arne
authors.initials: A
authors.lastname: Gennerich
mesh_terms: D002364:Caseins / Q000378:metabolism; D002465:Cell Movement; D055767:Immobilized Proteins; D008856:Microscopy, Fluorescence; D008870:Microtubules / Q000378:metabolism*; D020409:Molecular Motor Proteins / Q000378:metabolism*; D013194:Staining and Labeling; D014404:Tubulin / Q000378:metabolism
chemical_list: D002364:Caseins; D055767:Immobilized Proteins; D020409:Molecular Motor Proteins; D014404:Tubulin