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

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Mitosis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1136))

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.

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Acknowledgements

We thank Johan Andreasson, Robert Coleman, and Wei Chen for helpful discussions regarding aminosilanization and Laura E.K. Nicholas for assistance with photography and figure illustration. The authors are supported by the National Institutes of Health grant R01GM098469. M.P.N. received support from the NIH-funded Medical Scientist Training and Molecular Biophysics Training programs (NIH grants T32GM007288 and T32GM008572, respectively) at the Albert Einstein College of Medicine.

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  1. Department of Anatomy and Structural Biology, Gruss Lipper Biophotonics Center, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY, 10461, USA

    Matthew P. Nicholas, Lu Rao & Arne Gennerich

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  1. Matthew P. Nicholas
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  2. Lu Rao
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  3. Arne Gennerich
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Correspondence to Arne Gennerich .

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  1. Dept Physiology Biophysics, Albert Einstein College of Medicine, Bronx, New York, USA

    David J. Sharp

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Nicholas, M.P., Rao, L., Gennerich, A. (2014). Covalent Immobilization of Microtubules on Glass Surfaces for Molecular Motor Force Measurements and Other Single-Molecule Assays. In: Sharp, D. (eds) Mitosis. Methods in Molecular Biology, vol 1136. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0329-0_9

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