Department of Biochemistry and Molecular Biology, Mayo Clinic Rochester, Rochester, MN, 55905, USA.
Department of Biochemistry and Molecular Biology, Mayo Clinic Rochester, Rochester, MN, 55905, USA; Department of Physiology and Biomedical Engineering, Mayo Clinic Rochester, Rochester, MN, 55905, USA.
Anal Biochem. 2018 Dec 15;563:56-60. doi: 10.1016/j.ab.2018.10.007. Epub 2018 Oct 6.
The myosin motor powers cardiac contraction and is frequently implicated in hereditary heart disease by its mutation. Principal motor function characteristics include myosin unitary step size, duty cycle, and force-velocity relationship for translating actin under load. These characteristics are sometimes measured in vitro with a motility assay detecting fluorescent labeled actin filament gliding velocity over a planar array of surface immobilized myosin. Assay miniaturization in a polydimethylsiloxane/glass (PDMS/glass) hybrid microfluidic flow channel is an essential component to a small sample volume assay applicable to costly protein samples however the PDMS substrate dramatically inhibits myosin motility. Myosin in vitro motility in a PDMS/glass hybrid microfluidic flow cell was tested under a variety of conditions to identify and mitigate the effect of PDMS on myosin. Substantial contamination by unpolymerized species in the PDMS flow cells is shown to be the cause of myosin motility inhibition. Normal myosin motility recovers by either extended cell aging (~20 days) to allow more complete polymerization or by direct chemical extraction of the unpolymerized species from the polymer substrate. PDMS flow cell aging is the low cost alternative compatible with the other PDMS and glass modifications needed for in vitro myosin motility assaying.
肌球蛋白马达为心脏收缩提供动力，其突变常与遗传性心脏病有关。主要的运动功能特征包括肌球蛋白的单位步长、作用循环和在负载下转化肌动蛋白的力-速度关系。这些特征有时可以通过检测荧光标记的肌动蛋白丝在固定于表面的肌球蛋白平面阵列上滑动速度的运动分析来在体外测量。在聚二甲基硅氧烷/玻璃（PDMS/玻璃）混合微流控通道中的微缩化是适用于昂贵蛋白质样品的小样本量分析的重要组成部分，然而 PDMS 基质会极大地抑制肌球蛋白的运动。在各种条件下测试了 PDMS/玻璃混合微流控流动池中的肌球蛋白体外运动，以确定和减轻 PDMS 对肌球蛋白的影响。结果表明，PDMS 流动池中的未聚合物质的大量污染是肌球蛋白运动抑制的原因。通过延长细胞老化（~20 天）以允许更完全的聚合，或通过直接从聚合物基质中化学提取未聚合物质，正常的肌球蛋白运动可以恢复。PDMS 流动池老化是一种低成本的替代方案，与体外肌球蛋白运动分析所需的其他 PDMS 和玻璃改性兼容。
