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

1.非同步螺旋行星离心机分离蛋白质的研究采用非同步螺旋行星离心机，考察了螺旋柱的行星运动对蛋白质分离效率的影响，考察了螺旋离心机的最佳分离条件。分离采用3种不同体积(11、24和39mL)的螺旋柱，由12.5%(w/w)聚乙二醇1000和12.5%(w/w)磷酸二氢钾组成的两相系统和一组稳定的蛋白质样品(包括细胞色素C、肌红蛋白和溶菌酶)。在相同实验条件下，采用39mL多层螺旋多层螺旋在头到尾的洗脱模式下，转速为800rpm，旋转转速为10rpm时分离效果最佳，而偏心螺旋的分离效果与此类似。进一步的实验还表明，在头到尾的洗脱模式下，下流动相的顺时针(CW)线圈旋转或上流动相的逆时针(CCW)线圈旋转效果最好，这两种方式都是在转子的逆时针旋转下进行的。利用非同步CPC淋洗血细胞成分将非同步CPC应用于使用生理缓冲液分离血细胞成分。绵羊血样分离的最佳条件是采用头到尾的洗脱方式，在螺旋柱顺时针旋转的条件下进行分离。在600-1000rpm的高速转速下，羊和人的血液样本被完全分离并洗脱成两个峰，除红细胞外的所有细胞成分如血小板和白细胞以0rpm的速度洗脱，红细胞以10rpm的螺旋转速洗脱。羊血在800rpm转速下达到正常洗脱模式，人血样品在700rpm达到正常洗脱模式。

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.