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Magnetic Cytoskeleton Affinity Purification of Microtubule Motors Conjugated to Quantum Dots
Bioconjugate Chemistry ( IF 4.7 ) Pub Date : 2018-06-22 00:00:00 , DOI: 10.1021/acs.bioconjchem.8b00264
Marco Tjioe , Hyeon Ryoo , Yuji Ishitsuka , Pinghua Ge , Carol Bookwalter , Walter Huynh 1 , Richard J McKenney 2 , Kathleen M Trybus 3 , Paul R Selvin
Affiliation  

We develop magnetic cytoskeleton affinity (MiCA) purification, which allows for rapid isolation of molecular motors conjugated to large multivalent quantum dots, in miniscule quantities, which is especially useful for single-molecule applications. When purifying labeled molecular motors, an excess of fluorophores or labels is usually used. However, large labels tend to sediment during the centrifugation step of microtubule affinity purification, a traditionally powerful technique for motor purification. This is solved with MiCA, and purification time is cut from 2 h to 20 min, a significant time-savings when it needs to be done daily. For kinesin, MiCA works with as little as 0.6 μg protein, with yield of ∼27%, compared to 41% with traditional purification. We show the utility of MiCA purification in a force-gliding assay with kinesin, allowing, for the first time, simultaneous determination of whether the force from each motor in a multiple-motor system drives or hinders microtubule movement. Furthermore, we demonstrate rapid purification of just 30 ng dynein-dynactin-BICD2N-QD (DDB-QD), ordinarily a difficult protein-complex to purify.

中文翻译:

与量子点共轭的微管马达的磁性细胞骨架亲和纯化

我们开发了磁性细胞骨架亲和 (MiCA) 纯化,可以快速分离微量的与大型多价量子点缀合的分子马达,这对于单分子应用特别有用。当纯化标记的分子马达时,通常使用过量的荧光团或标记。然而,大的标签在微管亲和纯化的离心步骤中往往会沉淀,微管亲和纯化是一种传统的强大的运动纯化技术。MiCA 解决了这个问题,纯化时间从 2 小时缩短到 20 分钟,当需要每天进行时,可以节省大量时间。对于驱动蛋白,MiCA 只需 0.6 μg 蛋白质即可发挥作用,收率约为 27%,而传统纯化的收率为 41%。我们展示了 MiCA 纯化在驱动蛋白力滑动测定中的实用性,首次允许同时确定多电机系统中每个电机的力是否驱动或阻碍微管运动。此外,我们证明了仅 30 ng 动力蛋白-dynactin-BICD2N-QD (DDB-QD) 的快速纯化,这通常是一种难以纯化的蛋白质复合物。
更新日期:2018-06-22
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