Abstract
The purpose of this protocol is to provide an updated method of performing microtubule gliding assays and visualizing it using fluorescence microscopy.
Keywords: Gliding assay, Mitotic motor, in vitro motility, Polarity-marked microtubules, Glass chamber
Background
Mitotic spindles are protein machinery that dominate mitosis. The mitotic spindle utilizes microtubule-based motor proteins to organize itself, and exert forces to drive cell division. Microtubule-based motor proteins produce mechanical work using energy derived from ATP hydrolysis (Coppin et al., 1997). Motor proteins translocate microtubules in a unidirectional manner. The behavior of motility can be observed by in vitro gliding assay (Tao and Scholey, 2010), in which the motors are affixed onto a glass surface and supplied with microtubules and ATP. The motility of the motor proteins can then be studied using fluorescence microscopy and the details of their dynamic behavior can be observed in real time. This updated protocol will allow analysis of microtubule-based motor protein function with the use of in vitro microtubule gliding assays (Tao et al., 2006 and 2016).
Materials and Reagents
Equipment
Software
Procedure
Data analysis
Notes
Recipes
Acknowledgments
This method is adopted from Tao et al. (2016) and Tao et al. (2010). The above work was funded by NIH grant GM046409 to W.S. and NIH grant GM 55507 to J.M.S.
References
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.