Molecular motors, essential to force-generation and cargo transport within cells, are invaluable tools for powering nanobiotechnological lab-on-a-chip devices. These devices are based on in vitro motility assays that reconstitute molecular transport with purified motor proteins, requiring a deep understanding of the biophysical properties of motor proteins and thorough optimization to enable motility under varying environmental conditions. Until now, these assays have been prepared manually, severely limiting throughput. To overcome this limitation, we developed an in vitro motility assay where sample preparation, imaging and data evaluation are fully automated, enabling the processing of a 384-well plate within less than three hours. We demonstrate the automated assay for the analysis of peptide inhibitors for kinesin-1 at a wide range of concentrations, revealing that the IAK domain responsible for kinesin-1 auto-inhibition is both necessary and sufficient to decrease the affinity of the motor protein for microtubules, an aspect that was hidden in previous experiments due to scarcity of data.

中文翻译：

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用于分子马达高通量研究的 自动化体外运动测定

分子电动机对于细胞内的力产生和货物运输至关重要，是为纳米生物技术芯片实验室设备提供动力的宝贵工具。这些设备基于体外运动分析，该运动通过纯化的运动蛋白重构分子运输，需要深入了解运动蛋白的生物物理特性并进行全面优化以在不同的环境条件下实现运动。到目前为止，这些测定都是手动进行的，严重限制了通量。为了克服这一局限性，我们开发了一种体外全自动分析，其中样品制备，成像和数据评估是全自动的，可在不到三个小时的时间内处理384孔板。我们展示了在宽范围的浓度下分析用于kinesin-1的肽抑制剂的自动化分析方法，揭示了负责kinesin-1自动抑制的IAK结构域既必要又足以降低运动蛋白对微管的亲和力，由于缺乏数据，以前的实验中未显示此方面。