Prolongation of the active lifetime of a biomolecular motor for in vitro motility assay by using an inert atmosphere |
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Authors: | Kabir Arif Md Rashedul Inoue Daisuke Kakugo Akira Kamei Akiko Gong Jian Ping |
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Affiliation: | Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan. |
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Abstract: | Over the last few decades, the in vitro motility assay has been performed to probe the biophysical and chemo-mechanical properties as well as the self-organization process of biomolecular motor systems such as actin-myosin and microtubule-kinesin. However, aggression of the reactive oxygen species (ROS) and concomitant termination of the activity of biomolecular motors during investigation remains a drawback of this assay. Despite enzymatic protection that makes use of a combination of glucose, glucose oxidase, and catalase, the active lifetime of biomolecular motors is found to be only a few hours and this short lifetime restricts further study on those systems. We have solved this problem by using a newly developed system of the in vitro motility assay that is conducted in an inert nitrogen gas atmosphere free of ROS. Using microtubule-kinesin as a model system we have shown that our system has prolonged the active lifetime of the biomolecular motor until several days and even a week by protecting it from oxidative damage. |
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