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Methods Mol Biol. 2014;1136:171-246. doi: 10.1007/978-1-4939-0329-0_10.

An improved optical tweezers assay for measuring the force generation of single kinesin molecules.

Author information

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

Abstract

Numerous microtubule-associated molecular motors, including several kinesins and cytoplasmic dynein, produce opposing forces that regulate spindle and chromosome positioning during mitosis. The motility and force generation of these motors are therefore critical to normal cell division, and dysfunction of these processes may contribute to human disease. Optical tweezers provide a powerful method for studying the nanometer motility and piconewton force generation of single motor proteins in vitro. Using kinesin-1 as a prototype, we present a set of step-by-step, optimized protocols for expressing a kinesin construct (K560-GFP) in Escherichia coli, purifying it, and studying its force generation in an optical tweezers microscope. We also provide detailed instructions on proper alignment and calibration of an optical trapping microscope. These methods provide a foundation for a variety of similar experiments.

PMID:
24633799
PMCID:
PMC4254714
DOI:
10.1007/978-1-4939-0329-0_10
[Indexed for MEDLINE]
Free PMC Article

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