使用支撑脂质双层测量膜结合驱动蛋白马达的微管结合动力学。

Measuring microtubule binding kinetics of membrane-bound kinesin motors using supported lipid bilayers.

机构信息

Intercollege Program in Integrative and Biomedical Physiology, Pennsylvania State University, University Park, PA 16802, USA.

Department of Biomedical Engineering, Pennsylvania State University, University Park, PA 16802, USA.

出版信息

STAR Protoc. 2021 Jul 29;2(3):100691. doi: 10.1016/j.xpro.2021.100691. eCollection 2021 Sep 17.

DOI:10.1016/j.xpro.2021.100691

PMID:34382017

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC8339383/

Abstract

Membrane-bound cargos in cells are generally transported by multiple kinesin motors. Quantifying the bimolecular on-rate of motors for their microtubule track is important for understanding of multi-motor transport but is complicated by diffusion of the motors in the plane of the lipid bilayer. Here, we describe a method to measure the kinesin on-rate that uses a modified microtubule gliding assay performed on a supported lipid bilayer and detects motor binding by a local increase in fluorescence. For complete details on the use and execution of this protocol, please refer to Jiang et al. (2019).

摘要

细胞内的膜结合货物通常由多个驱动蛋白分子来运输。量化驱动蛋白与微管轨道的双分子结合速率对于理解多分子运输非常重要，但由于驱动蛋白在脂质双层平面内的扩散，这一过程变得复杂。在这里，我们描述了一种使用改良的在支撑脂质双层上进行的微管滑行测定法来测量驱动蛋白结合速率的方法，该方法通过荧光局部增加来检测马达的结合。有关此方案的使用和执行的完整详细信息，请参见 Jiang 等人。（2019 年）。