Development of Separation Methods for Biological Macromolecules and Cells Using Nonsynchronous Coil Planet Centrifuge

非同步线圈行星离心机生物大分子和细胞分离方法的发展

• 批准号: 14572039
• 负责人: SHINOMIYA Kazufusa
• 金额: $ 1.54万
• 依托单位: Nihon University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14572039/
• 关键词: countercurrent chromatoeraphy; aqueous two-phase partitioning; protein separation; cell separation; chromatosgraphy; separation analysis; biological macromolecule; blood cell; 水生二層分配法; 生態高分子

1. Protein separation using the nonsynchronous coil planet centrifuge (CPC)The nonsynchronous CPC was employed to investigate the optimal separating conditions and the effect of planetary motion of coiled column on the partition efficiency using the protein separation with aqueous-aqueous polymer phase systems. The separation was examined using three kinds coiled column with each different volume (11, 24 and 39 mL), a two-phase system composed of 12.5% (w/w) polyethylene glycol 1000 and 12.5% (w/w) dibasic potassium phosphate and a set of stable proteins for samples including cytochrome C, myoglobin and lysozyme. The best separation was obtained using the 39 mL multilayer coil at the head to tail elution mode with the revolution at 800 rpm and the rotation at 10 rpm, where the similar result was obtained in the eccentric coil under same experimental conditions. The further experiments also revealed that the best result was obtained in the head to tail elution mode by the clockwise (CW) coil rotation for the lower mobile phase or by the counterclockwise (CCW) coil rotation for the upper mobile phase, both under CCW revolution of the rotor.2. Elutriation of blood cell components by the nonsynchronous CPCThe nonsynchronous CPC was applied to the separation of blood cell components using a physiological buffer solution. The optimal condition for the separation of sheep blood samples was performed at the CCW revolution and the CW rotation of coiled column by the head to tail elution mode. Under the high speed of revolution of 600-1000 rpm, sheep and human blood samples were each completely separated and eluted into two peaks, where all cell components other than erythrocytes such as platelets and leukocytes were eluted at 0 rpm and erytbrocytes at 10 rpm of coil rotation. The normal elution pattern was achieved at 800 rpm of revolution for sheep blood and at 700 rpm for human blood samples.

1.非同步螺旋行星离心机（CPC）分离蛋白质研究了非同步CPC分离蛋白质的最佳分离条件以及螺旋柱的行星运动对蛋白质在水-水聚合物相体系中分配效率的影响。采用3种不同体积（11、24和39 mL）的螺旋柱，12.5%（w/w）的聚乙二醇1000和12.5%（w/w）的磷酸氢二钾组成的两相体系，以及一组稳定的蛋白质（细胞色素C、肌红蛋白和溶菌酶）进行分离。采用39 mL多层螺旋管在头-尾洗脱模式下，以800 rpm旋转和10 rpm旋转获得最佳分离，其中在相同的实验条件下，在偏心螺旋管中获得类似的结果。进一步的实验还表明，在头-尾洗脱模式下，对于下部移动的阶段，顺时针（CW）线圈旋转或对于上部移动的阶段，逆时针（CCW）线圈旋转均在转子逆时针旋转的情况下获得最佳结果.非同步CPC对血细胞组分的分离研究将非同步CPC应用于生理缓冲液中血细胞组分的分离。采用首尾洗脱方式，确定了螺旋柱逆时针旋转和顺时针旋转分离羊血样品的最佳条件。在600-1000 rpm的高速旋转下，绵羊和人血液样品各自完全分离并洗脱成两个峰，其中除红细胞以外的所有细胞组分如血小板和白细胞在0 rpm下洗脱，而红细胞在10 rpm的线圈旋转下洗脱。羊血在800 rpm转速下和人血样品在700 rpm转速下获得正常洗脱模式。

项目成果

Kazufusa Shinomiya et al.: "Protein separation by nonsynchronous coil planet centrifuge with aqueous-aqueous polymer phase systems"Journal of Chromatography A. 1005. 103-112 (2003)

Kazufusa Shinomiya 等人：“采用水-水聚合物相系统的非同步线圈行星式离心机进行蛋白质分离”Journal of Chromatography A. 1005. 103-112 (2003)

Kazufusa Shinomiya, Yozo Kabasawa, Kazuhiro Yanagidaira, Haruo Sasaki, Minoru Muto, Tadashi Okada, Yoichiro Ito: "Protein separation by nonsynchronous coil planet centrifuge with aqueous-aqueous polymer phase systems"Journal of Chromatography A. 1005. 103

Kazufusa Shinomiya、Yozo Kabasawa、Kazuhiro Yanagidaira、Haruo Sasaki、Minoru Muto、Tadashi Okada、Yoichiro Ito：“采用水-水聚合物相系统的非同步线圈行星离心机进行蛋白质分离”Journal of Chromatography A. 1005. 103